Krib polynucleotides and polypeptides and uses thereof in the treatment of metabolic disorders

ABSTRACT

The present invention relates to the field of metabolic research. KRIB polypeptides have been identified that are beneficial in the treatment of metabolic disorders. These compounds should be effective for reducing body mass and for treating metabolic disorders. These metabolic disorders include hyperlipidemia, atherosclerosis, diabetes, and hypertension.

FIELD OF THE INVENTION

The present invention relates to the field of metabolic research, inparticular the discovery of compounds effective for reducing body massand useful for treating metabolic disorders. The metabolic disordersenvisioned to be treated by the methods of the invention include, butare not limited to, hyperlipidemia, atherosclerosis, diabetes, andhypertension.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

KRIB Polypeptides

KRIB polypeptides are secreted proteins of human origin. KRIB-1 is asecreted cytokine-like protein, the full-length protein being 136 aminoacids long (EMBL Accession No. NP_(—)061129). KRIB-1 is expressed inCD31+ hematopoietic stem cells (Liu et al., Genomics 2000; 65:283-92),and has been shown to drive the proliferation of mesenchymal stem cells(WO 00/63382). KRIB-4 is a secreted protein also named leucine-richalpha 2-glycoprotein (EMBL Eccession No. NP_(—)443204). The structuralcharacteristics of KRIB-4 suggest that it may be a membrane-derived ormembrane-associated protein containing a series of domains capable ofbipolar surface orientation (Takahashi et al., Proc Natl Acad. Sci.1985; 82: 1906-10). It has been suggested that KRIB-4 may interact withthe TGF-beta 1 receptor (Sun et al., Cancer Lett 1995; 89:73-9). KRIB-2,KRIB-2R and KRIB-3 are poorly characterized. The full-length KRIB-2protein, encoded by GeneSeqN Accession No. AAX30395, is 97 amino acidslong. KRIB-2R is encoded by EMBL Accession No. XM_(—)092166, and theKRIB-3 polypeptide, also named decidual protein induced by progesterone,corresponds to EMBL Accession No. NP_(—)008952.

Obesity and Related Metabolic Disorders

Obesity is a public health problem that is serious, widespread, andincreasing. In the United States, 20% of the population is obese; inEurope, a slightly lower percentage is obese (Friedman (2000) Nature404:632-634). In obese patients, the accumulation of excess fat putspressure on the lungs, causing difficulty in breathing and shortness ofbreath. The difficulty in breathing seriously interferes with sleep,causing momentary cessation of breathing leading to daytime sleepinessand other complications. Obesity also leads to various orthopedicproblems, including low back pain and worsening of osteoarthritis.Moreover, obesity is associated with increased risk of hypertension,cardiovascular disorder, diabetes, and cancer (Kopelman (2000) Nature404:635-643). Even modest weight loss ameliorates these associatedconditions.

Maintenance of weight gain or loss is associated with compensatorychanges in energy expenditure that oppose the maintenance of a bodyweight that is different from the usual weight [Leibel et al. (1995) NEngl J Med 332:621-8]. These changes may account, in part, for the poorlong-term efficacy of obesity treatments [Wadden (1993) Ann Intern Med229:688-93]. Further, the decreased insulin sensitivity after weightgain and the beneficial effects of even modest amounts of weightreduction on carbohydrate metabolism and insulin sensitivity in somepatients are well documented [Olefsky et al. (1974) J Clin Invest53:64-76].

While still acknowledging that lifestyle factors including environment,diet, age and exercise play a role in obesity, twin studies, analyses offamilial aggregation, and adoption studies all indicate that obesity islargely the result of genetic factors (Barsh et al (2000) Nature404:644-651). In agreement with these studies, is the fact that anincreasing number of metabolic genes are being identified. Some of themore extensively studied genes include those encoding leptin (ob) andits receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor(Mc4r), agouti protein (As), carboxypeptidase E (fat),5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubby protein (tubby)(rev'd in Barsh et al (2000) Nature 404:644-651).

Obesity is a risk factor for several metabolic disorders, and especiallyfor non-insulin-dependent (type II) diabetes. There is a strongcorrelation between these two disorders since 80 to 90% ofnon-insulin-dependent diabetic people are obese. Type II diabetes is acommon disorder, and about 15% of people over age 70 suffer from type IIdiabetes. In type II diabetes, the body develops resistance to theeffects of insulin, resulting in a relative insulin deficiency.Increased urination and thirst are the first symptoms. When the bloodsugar level becomes very high, the patient may develop severedehydration, which may lead to mental confusion, drowsiness, seizures,and hyperglycemic coma.

Furthermore, people with diabetes may suffer from many serious long-termcomplications. These complications notably encompass, e.g., heartattacks and strokes, loss of vision due to damage to the blood vesselsof the eye, kidney failure resulting in a dialysis requirement, and poorblood supply to the skin that can lead to ulcers and amputation in theworst cases.

Accordingly, obesity and related metabolic disorders such as insulinresistance and type II diabetes are a striking health problem. There iscurrently a need of novel and efficient drugs being capable of treatingobesity and preventing related metabolic disorders.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods of treating orpreventing metabolic disorders such as obesity. More specifically, thepresent invention is directed to pharmaceutical compositions comprisingKRIB polypeptides or fragments thereof, and to methods of using KRIBpolypeptides and fragments thereof for treating obesity. The presentinvention stems from the finding that KRIB polypeptides arecharacterized by elevated expression in human adipose tissue. Applicantshave found that KRIB polypeptides of the present invention have noveland unexpected utility for the treatment of insulin resistance,diabetes, and/or obesity, as well as disorders associated therewith. TheKRIB polypeptides of the present invention are secreted polypeptidesthat are able to lower circulating (either blood, serum, or plasma)levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or(iii) triglycerides. KRIB polypeptide are further able to (i) preventweight gain, (ii) reduce weight, and/or (iii) maintain weight loss.

The invention includes polypeptides encoded by KRIB-1, which includeboth the full-length polypeptide and fragments thereof. As used herein,a “KRIB-1 polypeptide” refers to a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or fragments and variants thereof. The KRIB-1polypeptide has in vitro and in vivo biological activity as describedherein, including utility for weight reduction, prevention of weightgain and control of blood glucose levels in humans and other mammals.

The invention includes polypeptides encoded by KRIB-2 and KRIB-2R, whichinclude both the full-length polypeptides and fragments thereof. Saidpolypeptide fragments may comprise all or part of the LAWN domain. Asused herein, a “KRIB-2 polypeptide” refers to a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 4 or fragments and variantsthereof. As used herein, “a KRIB-2R polypeptide” refers to a polypeptidecomprising the amino acid sequence of SEQ ID NO: 6 or fragments andvariants thereof. Preferably, the KRIB-2 and KRIB-2R polypeptidescontain all or part of the LAWN domain and have in vitro and in vivobiological activity as described herein, including utility for weightreduction, prevention of weight gain and control of blood glucose levelsin humans and other mammals.

The invention includes polypeptides encoded by KRIB-3, which includeboth the full-length polypeptide and fragments thereof. As used herein,“a KRIB-3 polypeptide” refers to a polypeptide comprising the amino acidsequence of SEQ ID NO: 8 or fragments and variants thereof. The KRIB-3polypeptide has in vitro and in vivo biological activity as describedherein, including utility for weight reduction, prevention of weightgain and control of blood glucose levels in humans and other mammals.

The invention includes polypeptides encoded by KRIB-4. The KRIB-4full-length polypeptide is comprised of (i) eight Leucine-rich repeats(LRRs); (ii) one Leucine rich repeat C-terminal domain (LRRCT); (iii)eight segments The polypeptides encoded by KRIB-4 include both thefull-length polypeptide and fragments thereof, said polypeptidefragments preferably comprising all or part of (i) one of the LRRdomains; (ii) one of the segments exhibiting a periodic pattern in theoccurrence of leucine, proline, and asparagines; or (iii) the LRRCTdomain. As used herein, a “KRIB-4 polypeptide” refers to a polypeptideof SEQ ID NO: 10 or fragments and variants thereof. Preferably, theKRIB-4 polypeptides comprise all or part of (i) one of the LRR domains;(ii) one of the segment exhibiting a periodic pattern in the occurrenceof leucine, proline, and asparagines; and/or (iii) the LRRCT domain, andhave in vitro and in vivo biological activity as described herein,including utility for weight reduction, prevention of weight gain andcontrol of blood glucose levels in humans and other mammals.

As used herein, the term “KRIB polypeptide” refers to a polypeptideselected from the group consisting of a KRIB-1 polypeptide, a KRIB-2polypeptide, a KRIB-2R polypeptide, a KRIB-3 polypeptide and a KRIB-4polypeptide.

More specifically, the biological activities of the KRIB polypeptides,including fragments, include reduction of elevated free fatty acidlevels caused by administration of epinephrine, i.v. injection of“intralipid”, or administration of a high fat test meal, as well asincreased fatty acid oxidation in muscle cells, reduction in glucoselevels, modulation of energy expenditure, prevention and/or reduction ofinsulin resistance, and weight reduction in mammals consuming a highfat/high sucrose diet.

Thus, the invention is drawn to KRIB polypeptides, polynucleotidesencoding said KRIB polypeptides, vectors comprising said KRIBpolynucleotides, and cells recombinant for said KRIB polynucleotides, aswell as to pharmaceutical and physiologically acceptable compositionscomprising said KRIB polypeptides and methods of administering said KRIBpharmaceutical and physiologically acceptable compositions in order toreduce body weight or to treat metabolic disorders. Assays foridentifying agonists and antagonists of metabolic activity are also partof the invention.

Antagonists of KRIB polypeptide activity should be effective in thetreatment of other metabolic disorders of the invention includingcachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia. In preferred embodiments, said individual is amammal, preferably a human.

In a first aspect, the invention features purified, isolated, orrecombinant KRIB polypeptides that have lipid partitioning, lipidmetabolism, and insulin-like activities. Preferred KRIB polypeptidefragments are said polypeptide fragments having activity, wherein saidactivity is also selected from the group consisting of lipidpartitioning, lipid metabolism, and insulin-like activity. In preferredembodiments, said polypeptide fragment comprises, consists essentiallyof, or consists of, at least 6 consecutive amino acids and not more than136 consecutive amino acids of SEQ ID NO: 2. In preferred embodiments,said polypeptide fragment comprises, consists essentially of, orconsists of, at least 6 consecutive amino acids and not more than 97consecutive amino acids of SEQ ID NO: 4 or 111 consecutive amino acidsof SEQ ID NO: 6. In preferred embodiments, said polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids and not more than 212 consecutive amino acids ofSEQ ID NO: 8. In preferred embodiments, said polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids and not more than 347 consecutive amino acids ofSEQ ID NO: 10.

In other preferred embodiments, KRIB-1 polypeptide fragments havingactivity are selected from amino acids 20-136, 21-136, 22-136, 23-136,24-136, 25-136, 26-136, 27-136, 28-136, 29-136, 30-136, 31-136, 32-136,33-136, 34-136, 35-136, 36-136, 37-136, 38-136, 39-136, 40-136, 41-136,42-136, 43-136, 44-136, 45-136, 46-136, 47-136, 48-136, 49-136, 50-136,51-136, 52-136, 53-136, 54-136, 55-136, 56-136, 57-136, 58-136, 59-136,60-136, 61-136, 62-136, 63-136, 64-136, 65-136, 66-136, 67-136, 68-136,69-136, 70-136, 71-136, 72-136, 73-136, 74-136, 75-136, 76-136, 77-136,78-136, 79-136, 80-136, 81-136, 82-136, 83-136, 84-136, 85-136, 86-136,87-136, 88-136, 89-136, 90-136, 91-136, 92-136, 93-136, 94-136, 95-136,96-136, 97-136, 98-136, 99-136, 100-136, 101-136, 102-136, 103-136,104-136, 105-136, 106-136, 107-136, 108-136, 109-136, 110-136, 111-136,112-136, 113-136, 114-136, 115-136, 116-136, 117-136, 118-136, 119-136,120-136, 121-136, 122-136, 123-136, 124-136, 125-136, 126-136, 127-136,128-136, 129-136 or 130-136 of SEQ ID NO: 2, where it is understood thatamino acid 20 is taken to represent the N-terminal amino acid of matureKRIB-1 polypeptide absent the signal peptide.

In other preferred embodiments, KRIB-2 polypeptide fragments havingactivity are selected from amino acids 15-97, 16-97, 17-97, 18-97,19-97, 20-97, 21-97, 22-97, 23-97, 24-97; 25-97, 26-97, 27-97, 28-97,29-97, 30-97, 31-97, 32-97, 33-97, 34-97, 35-97, 36-97, 37-97, 38-97,39-97, 40-97, 41-97, 42-97, 43-97, 44-97, 45-97, 46-97, 47-97, 48-97,49-97, 50-97, 51-97, 52-97, 53-97, 54-97, 55-97, 56-97, 57-97, 58-97,59-97, 60-97, 61-97, 62-97, 63-97, 64-97, 65-97, 66-97, 67-97, 68-97,69-97, 70-97, 71-97, 72-97, 73-97, 74-97, 75-97, 76-97, 77-97, 78-97,79-97, 80-97, 81-97, 82-97, 83-97, 84-97, 85-97, 86-97, 87-97, 88-97,89-97, 90-97, or 91-97 of SEQ ID NO: 4, where it is understood thatamino acid 15 is taken to represent the N-terminal amino acid of matureKRIB-2 polypeptide absent the signal peptide.

In other preferred embodiments, KRIB-2R polypeptide fragments havingactivity are selected from amino acids 15-111, 16-111, 17-111, 18-111,19-111, 20-111, 21-111, 22-111, 23-111, 24-111, 25-111, 26-111, 27-111,28-111, 29-111, 30-111, 31-111, 32-111, 33-111, 34-111, 35-111, 36-111,37-111, 38-111, 39-111, 40-111, 41-111, 42-111, 43-111, 44-111, 45-111,46-111, 47-111, 48-111, 49-111, 50-111, 51-111, 52-111, 53-111, 54-111,55-111, 56-111, 57-111, 58-111, 59-111, 60-111, 61-111, 62-111, 63-111,64-111, 65-111, 66-111, 67-111, 68-111, 69-111, 70-111, 71-111, 72-111,73-111, 74-111, 75-111, 76-111, 77-111, 78-111, 79-111, 80-111, 81-111,82-111, 83-111, 84-111, 85-111, 86-111, 87-111, 88-111, 89-111, 90-111,91-111, 92-111, 93-111, 94-111, 95-111, 96-111, 97-111, 98-111, 99-111,100-111, 101-111, 102-111, 103-111, 104-111, or 105-111 of SEQ ID NO: 6,where it is understood that amino acid 15 is taken to represent theN-terminal amino acid of mature KRIB-2R polypeptide absent the signalpeptide.

In other preferred embodiments, KRIB-3 polypeptide fragments havingactivity are selected from amino acids 18-212, 19-212, 20-212, 21-212,22-212, 23-212, 24-212, 25-212, 26-212, 27-212, 28-212, 29-212, 30-212,31-212, 32-212, 33-212, 34-212, 35-212, 36-212, 37-212, 38-212, 39-212,40-212, 41-212, 42-212, 43-212, 44-212, 45-212, 46-212, 47-212, 48-212,49-212, 50-212, 51-212, 52-212, 53-212, 54-212, 55-212, 56-212, 57-212,58-212, 59-212, 60-212, 61-212, 62-212, 63-212, 64-212, 65-212, 66-212,67-212, 68-212, 69-212, 70-212, 71-212, 72-212, 73-212, 74-212, 75-212,76-212, 77-212, 78-212, 79-212, 80-212, 81-212, 82-212, 83-212, 84-212,85-212, 86-212, 87-212, 88-212, 89-212, 90-212, 91-212, 92-212, 93-212,94-212, 95-212, 96-212, 97-212, 98-212, 99-212, 100-212, 101-212,102-212, 103-212, 104-212, 105-212, 106-212, 107-212, 108-212, 109-212,110-212, 111-212, 112-212, 113-212, 114-212, 115-212, 116-212, 117-212,118-212, 119-212, 120-212, 121-212, 122-212, 123-212, 124-212, 125-212,126-212, 127-212, 128-212, 129-212, 130-212, 131-212, 132-212, 133-212,134-212, 135-212, 136-212, 137-212, 138-212, 139-212, 140-212, 141-212,142-212, 143-212, 144-212, 145-212, 146-212, 147-212, 148-212, 149-212,150-212, 151-212, 152-212, 153-212, 154-212, 155-212, 156-212, 157-212,158-212, 159-212, 160-212, 161-212, 162-212, 163-212, 164-212, 165-212,166-212, 167-212, 168-212, 169-212, 170-212, 171-212, 172-212, 173-212,174-212, 175-212, 176-212, 177-212, 178-212, 179-212, 180-212, 181-212,182-212, 183-212, 184-212, 185-212, 186-212, 187-212, 188-212, 189-212,190-212, 191-212, 192-212, 193-212, 194-212, 195-212, 196-212, 197-212,198-212, 199-212, 200-212, 201-212, 202-212, 203-212, 204-212, 205-212,206-212 or 207-212 of SEQ ID NO: 8, where it is understood that aminoacid 18 is taken to represent the N-terminal amino acid of mature KRIB-3polypeptide absent the signal peptide.

In other preferred embodiments, KRIB-4 polypeptide fragments havingactivity are selected from amino acids 36-347, 37-347, 38-347, 39-347,40-347, 41-347, 42-347, 43-347, 44-347, 45-347, 46-347, 47-347, 48-347,49-347, 50-347, 51-347, 52-347, 53-347, 54-347, 55-347, 56-347, 57-347,58-347, 59-347, 60-347, 61-347, 62-347, 63-347, 64-347, 65-347, 66-347,67-347, 68-347, 69-347, 70-347, 71-347, 72-347, 73-347, 74-347, 75-347,76-347, 77-347, 78-347, 79-347, 80-347, 81-347, 82-347, 83-347, 84-347,85-347, 86-347, 87-347, 88-347, 89-347, 90-347, 91-347, 92-347, 93-347,94-347, 95-347, 96-347, 97-347, 98-347, 99-347, 100-347, 101-347,102-347, 103-347, 104-347, 105-347, 106-347, 107-347, 108-347, 109-347,110-347, 111-347, 112-347, 113-347, 114-347, 115-347, 116-347, 117-347,118-347, 119-347, 120-347, 121-347, 122-347, 123-347, 124-347, 125-347,126-347, 127-347, 128-347, 129-347, 130-347, 131-347, 132-347, 133-347,134-347, 135-347, 136-347, 137-347, 138-347, 139-347, 140-347, 141-347,142-347, 143-347, 144-347, 145-347, 146-347, 147-347, 148-347, 149-347,150-347, 151-347, 152-347, 153-347, 154-347, 155-347, 156-347, 157-347,158-347, 159-347, 160-347, 161-347, 162-347, 163-347, 164-347, 165-347,166-347, 167-347, 168-347, 169-347, 170-347, 171-347, 172-347, 173-347,174-347, 175-347, 176-347, 177-347, 178-347, 179-347, 180-347, 181-347,182-347, 183-347, 184-347, 185-347, 186-347, 187-347, 188-347, 189-347,190-347, 191-347, 192-347, 193-347, 194-347, 195-347, 196-347, 197-347,198-347, 199-347, 243-347, 244-347, 245-347, 246-347, 247-347, 248-347,249-347, 250-347, 251-347, 252-347, 253-347, 254-347, 255-347, 256-347,257-347, 258-347, 259-347, 260-347, 261-347, 262-347, 263-347, 264-347,265-347, 266-347, 267-347, 268-347, 269-347, 270-347, 271-347, 272-347,273-347, 274-347, 275-347, 276-347, 277-347, 278-347, 279-347, 280-347,281-347, 282-347, 283-347, 284-347, 285-347, 286-347, 287-347, 288-347,289-347, 290-347, 291-347, 292-347, 293-347, 294-347, 295-347, 296-347,297-347, 298-347, 299-347, 300-347, 301-347, 302-347, 303-347, 304-347,305-347, 306-347, 307-347, 308-347, 309-347, 110-347, 311-347, 312-347,313-347, 314-347, 315-347, 316-347, 317-347, 318-347, 319-347, 320-347,321-347, 322-347, 323-347, 324-347, 325-347, 326-347, 327-347, 328-347,329-347, 330-347, 331-347, 332-347, 333-347, 334-347, 335-347, 336-347,337-347, 338-347, 339-347, 340-347, 341-347 or 342-347 of SEQ ID NO: 10,where it is understood that amino acid 36 is taken to represent theN-terminal amino acid of mature KRIB-4 polypeptide absent the signalpeptide.

In further preferred embodiments, said polypeptide fragment comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the correspondingconsecutive amino acids of the polypeptide sequences identified in evenSEQ ID NOs: 2-10.

The invention further provides purified, isolated, or recombinant KRIBpolypeptides that have weight reduction activities. Preferred KRIBpolypeptide fragments are said polypeptide fragments having activity,wherein said activity is also selected from the group consisting ofprevention of weight gain, weight reduction, and maintenance of weightloss.

In preferred embodiments, said KRIB-1 polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 136 consecutive amino acids of SEQ ID NO: 2. Inpreferred embodiments, said KRIB-2 polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 97 consecutive amino acids of SEQ ID NO: 4. Inpreferred embodiments, said KRIB-2R polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 111 consecutive amino acids of SEQ ID NO: 6. Inpreferred embodiments, said KRIB-3 polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 212 consecutive amino acids of SEQ ID NO: 8. Inpreferred embodiments, said KRIB-4 polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 347 consecutive amino acids of SEQ ID NO: 10.

The invention yet further provides a purified or isolated polypeptidecomprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of: (a) a full-length KRIBpolypeptide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding polypeptide of even SEQ ID NOs: 2-10; (b)a full-length KRIB polypeptide at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the corresponding polypeptide of evenSEQ ID NOs: 2-10; (c) a full-length KRIB polypeptide of even SEQ ID NOs:2-10 absent the N-terminal Met; (d) a mature KRIB polypeptide of evenSEQ ID NOs: 2-10 lacking signal peptide; (e) a KRIB polypeptide of evenSEQ ID NOs: 2-10 wherein said KRIB polypeptide is of any one integer inlength between 6 amino acids and 136, 97, 111, 212 and 347 amino acidsrespectively inclusive of even SEQ ID NOs: 2-10, respectively; (f) theepitope-bearing fragments of a KRIB polypeptide of even SEQ ID NOs:2-10; (g) the allelic variant polypeptides of any of the polypeptides of(a)-(f). The invention further provides for fragments of thepolypeptides of (a)-(g) above, such as those having biological activityor comprising biologically functional domain(s).

In other highly preferred embodiments, KRIB-2 and KRIB-2R polypeptidescomprise, consist essentially of, or consist of, a purified, isolated,or a recombinant KRIB-2 and KRIB-2R polypeptide fragment comprised ofall or part of the LAWN domain. Preferably, said KRIB-2 and KRIB-2Rpolypeptide fragment comprises, consists essentially of, or consists of,at least 6 consecutive amino acids of amino acids 15-97 of SEQ ID NO: 4or of amino acids 15-111 of SEQ ID NO: 6, where it is understood thatamino acid 15 is taken to represent the N-terminal amino acid of matureKRIB-2 and KRIB-2R polypeptide absent the signal peptide. Mostpreferably, said KRIB-2 and KRIB-2R polypeptide fragments havingactivity selected from the group consisting of prevention of weightgain, weight reduction, and maintenance of weight loss comprise the LAWNDomain region about from amino acids 72-94 of SEQ ID NO: 4 or from aboutamino acids 39-61 of SEQ ID NO: 6.

In one highly preferred aspect, said KRIB-4 polypeptide fragmentscomprise all or part of one of the LRR domains. Preferably, said KRIB-4polypeptide fragments comprising all or part of one of the LRR domainsare selected from amino acids 36-347, 36-116, 36-140, 36-164, 36-188,36-212, 36-236, 36-260, 36-284, 93-116, 117-140, 141-164, 165-188,189-212, 213-236, 237-260, 261-284, 93-140, 93-164, 93-188, 93-212,93-236, 93-260, 93-284, 117-164, 117-188, 117-212, 117-236, 117-260,117-284, 141-188, 141-212, 141-236, 141-260, 141-284, 165-212, 165-236,165-260, 165-284, 189-236, 189-260, 189-284, 213-260, 213-284 and237-284 of SEQ ID NO: 10, where it is understood that amino acid 36 istaken to represent the N-terminal amino acid of mature KRIB-4polypeptide absent the signal peptide.

In another highly preferred aspect, said KRIB-4 polypeptide fragmentscomprise all or part of one of the segments exhibiting a periodicpattern in the occurrence of leucine, proline, and asparagines.Preferably, said KRIB-4 polypeptide fragments comprising all or part ofone of the segments exhibiting a periodic pattern in the occurrence ofleucine, proline, and asparagines are selected from amino acids 36-347,36-107, 36-131, 36-155, 36-179, 36-203, 36-227, 36-251, 36-275, 84-107,109-131, 132-155, 156-179, 180-203, 204-227, 228-251, 252-275, 84-131,84-155, 84-179, 84-203, 84-227, 84-251, 84-275, 109-155, 109-179,109-203, 109-227, 109-251, 109-275, 132-179, 132-203, 132-227, 132-251,132-275, 156-203, 156-227, 156-251, 156-275, 180-227, 180-251, 180-275,204-251, 204-275 and 228-275 of SEQ ID NO: 10, where it is understoodthat amino acid 36 is taken to represent the N-terminal amino acid ofmature KRIB-4 polypeptide absent the signal peptide.

In still another highly preferred aspect, said KRIB-4 polypeptidefragments comprise all or part of the LRRCT domain. Preferably, saidKRIB-4 polypeptide fragments comprising all or part of the LRRCT domainare selected from amino acids 36-347, 299-347, 93-347, 117-347, 141-347,165-347, 189-347, 213-347, 237-347, 261-347, 84-347, 109-347, 132-347,156-347, 180-347, 204-347, 228-347 and 252-347 of SEQ ID NO: 10, whereit is understood that amino acid 36 is taken to represent the N-terminalamino acid of mature KRIB-4 polypeptide absent the signal peptide.

In other highly preferred embodiments, KRIB-1 polypeptides comprise,consist essentially of, or consist of, a purified, isolated, or arecombinant KRIB-1 polypeptide fragment and having ability to bind tocells and thereby lead to dephosphorylation of protein kinase C alphawithin said cells. Preferably, said KRIB-1 polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 20-136, 21-136, 22-136, or 23-136of SEQ ID NO: 2, where it is understood that amino acid 20 is taken torepresent the N-terminal amino acid of mature KRIB-1 polypeptide absentthe signal peptide. In other highly preferred embodiments, KRIB-2 andKRIB-2R polypeptides comprise, consist essentially of, or consist of, apurified, isolated, or a recombinant KRIB-2 and KRIB-2R polypeptidefragment having ability to bind to cells and thereby lead todephosphorylation of protein kinase C alpha within said cells.Preferably, said KRIB-2 and KRIB-2R polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids of amino acids 15-97 of SEQ ID NO: 2 or 15-111 of SEQ ID NO: 4 andSEQ ID NO: 6, where it is understood that amino acid 15 is taken torepresent the N-terminal amino acid of mature KRIB-2 and KRIB-2Rpolypeptide absent the signal peptide. More preferably, KRIB-2 andKRIB-2R polypeptide fragments leading to dephosphorylation of proteinkinase C alpha are further comprised of all or part of the LAWN domain.In other highly preferred embodiments, KRIB-3 polypeptides comprise,consist essentially of, or consist of, a purified, isolated, or arecombinant KRIB-3 polypeptide fragment and having ability to bind tocells and thereby lead to dephosphorylation of protein kinase C alphawithin said cells. Preferably, said KRIB-3 polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 18-212 of SEQ ID NO: 8, where itis understood that amino acid 18 is taken to represent the N-terminalamino acid of mature KRIB-3 polypeptide absent the signal peptide. Inother highly preferred embodiments, KRIB-4 polypeptides comprise,consist essentially of, or consist of, a purified, isolated, or arecombinant KRIB-4 polypeptide fragment and having ability to bind tocells and thereby lead to dephosphorylation of protein kinase C alphawithin said cells. Preferably, said KRIB-4 polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 36-347 of SEQ ID NO: 10, where itis understood that amino acid 36 is taken to represent the N-terminalamino acid of mature KRIB-4 polypeptide absent the signal peptide. Morepreferably, KRIB-4 polypeptide fragments leading to dephosphorylation ofprotein kinase C alpha are further comprised of all or part of (i) oneof the LRR domains; (ii) one of the segment exhibiting a periodic patterin the occurrence of leucine, proline, and asparagines; and/or (iii) theLRRCT domain.

In other highly preferred embodiments, KRIB-1 polypeptides comprise,consist essentially of, or consist of, a purified, isolated, or arecombinant KRIB-1 polypeptide fragment and having ability to bind tocells and thereby lead to activation of NF-kB within said cells.Preferably, said KRIB-1 polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids ofamino acids 20-136, 21-136, 22-136, or 23-136 of SEQ ID NO: 2, where itis understood that amino acid 20 is taken to represent the N-terminalamino acid of mature KRIB-1 polypeptide absent the signal peptide. Inother highly preferred embodiments, KRIB-2 and KRIB-2R polypeptidescomprise, consist essentially of, or consist of, a purified, isolated,or a recombinant KRIB-2 and KRIB-2R polypeptide fragment having abilityto bind to cells and thereby lead to activation of NF-kB within saidcells. Preferably, said KRIB-2 and KRIB-2R polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 15-97 of SEQ ID NO: 4 or 15-111of SEQ ID NO: 6, where it is understood that amino acid 15 is taken torepresent the N-terminal amino acid of mature KRIB-2 and KRIB-2Rpolypeptide absent the signal peptide. More preferably, KRIB-2 andKRIB-2R polypeptide fragments leading to activation of NF-kB are furthercomprised of all or part of the LAWN domain. In other highly preferredembodiments, KRIB-3 polypeptides comprise, consist essentially of, orconsist of, a purified, isolated, or a recombinant KRIB-3 polypeptidefragment and having ability to bind to cells and thereby lead toactivation of NF-κB within said cells. Preferably, said KRIB-3polypeptide fragment comprises, consists essentially of, or consists of,at least 6 consecutive amino acids of amino acids 18-212 of SEQ ID NO:8, where it is understood that amino acid 18 is taken to represent theN-terminal amino acid of mature KRIB-4 polypeptide absent the signalpeptide. In other highly preferred embodiments, KRIB-4 polypeptidescomprise, consist essentially of, or consist of, a purified, isolated,or a recombinant KRIB-4 polypeptide fragment and having ability to bindto cells and thereby lead to activation of NF-κB within said cells.Preferably, said KRIB-4 polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids ofamino acids 36-347 of SEQ ID NO: 10, where it is understood that aminoacid 36 is taken to represent the N-terminal amino acid of mature KRIB-4polypeptide absent the signal peptide. More preferably, KRIB-4polypeptide fragments leading to activation of NF-κB are furthercomprised of all or part of (i) one of the LRR domains; (ii) one of thesegment exhibiting a periodic pattern in the occurrence of leucine,proline, and asparagines; and/or (iii) the LRRCT domain.

In a further preferred embodiment, KRIB polypeptides are able to lowercirculating (either in blood, serum or plasma) levels (concentration)of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.Further preferred polypeptides of the invention demonstrating free fattyacid level lowering activity, glucose level lowering activity, and/ortriglyceride level lowering activity, have an activity that is the sameor greater than full-length KRIB polypeptides at the same molarconcentration, have the same or greater than transient activity and/orhave a sustained activity.

In other further preferred embodiment, KRIB polypeptides are able toprevent weight gain, reduce weight, and/or maintain weight loss. Furtherpreferred polypeptides of the invention demonstrating prevention ofweight gain, weight reduction, and/or maintenance of weight loss, havean activity that is the same or greater than full-length KRIBpolypeptides at the same molar concentration, have the same or greaterthan transient activity and/or have a sustained activity.

Further preferred KRIB polypeptides are those that maintain weight loss,preferably in individuals who previously were “obese” and are now“healthy” (as defined herein).

Further preferred KRIB polypeptides are those that significantlystimulate muscle lipid or free fatty acid oxidation. Further preferredKRIB polypeptides are those that significantly stimulate muscle lipid orfree fatty acid oxidation.

Further preferred KRIB polypeptides are those that cause C2C12 cellsdifferentiated in the presence of said polypeptides to undergo at least10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared tountreated cells.

Further preferred KRIB polypeptides are those that increase leptinuptake in a liver cell line (preferably BPRCL mouse liver cells (ATCCCRL-2217)).

Further preferred KRIB polypeptides are those that significantly reducethe postprandial increase in plasma free fatty acids due to a high fatmeal.

Further preferred KRIB polypeptides are those that significantly reduceor eliminate ketone body production as the result of a high fat meal.

Further preferred KRIB polypeptides are those that increase glucoseuptake in skeletal muscle cells.

Further preferred KRIB polypeptides are those that increase glucoseuptake in adipose cells.

Further preferred KRIB polypeptides are those that increase glucoseuptake in neuronal cells.

Further preferred KRIB polypeptides are those that increase glucoseuptake in red blood cells.

Further preferred KRIB polypeptides are those that increase glucoseuptake in the brain.

Further preferred KRIB polypeptides are those that significantly reducethe postprandial increase in plasma glucose following a meal,particularly a high carbohydrate meal.

Further preferred KRIB polypeptides are those that significantly preventthe postprandial increase in plasma glucose following a meal,particularly a high fat or a high carbohydrate meal.

Further preferred KRIB polypeptides are those that increase insulinsensitivity.

Further preferred KRIB polypeptides are those that inhibit theprogression from impaired glucose tolerance to insulin resistance.

Further preferred KRIB polypeptides are those that form multimers (e.g.,heteromultimers or homomultimers) in vitro and/or in vivo. Preferredmultimers are homotrimers or homohexamers. Other preferred multimers arehomomultimers comprising at least 2, 3, 4, 6, 8, 9, 10 or 12 KRIBpolypeptide subunits. Further preferred multimers are heterotrimers orheterohexamers. Other preferred multimers are heteromultimers comprisingat least 1, 2, 3, 4, 6, 8, 9, 10 or 12 KRIB polypeptide subunits. Otherpreferred KRIB polypeptides are those that form multimers in vitroand/or in vivo that are able to bind to cells and thereby lead todephosphorylation of protein kinase C alpha within said cells. Otherpreferred KRIB polypeptides are those that form multimers in vitroand/or in vivo that are able to bind to cells and thereby lead toactivation of NF-kB within said cells.

Further preferred embodiments include heterologous polypeptidescomprising one of the KRIB polypeptides of the invention. More preferredis said heterologous polypeptide comprised of a signal peptide fused tothe N-terminus of said KRIB polypeptide of the invention. In yet morepreferred embodiment, said signal peptide is human zinc-alpha2-glycoprotein signal peptide of amino acid sequenceMVRMVPVLLSLLLLLGPAVP, preferably encoded by the polynucleotide ofsequence atggtaagaatggtgcctgtcctgctgtctctgctgctgcttctgggtcctgctgtcccc.

In a second aspect, the invention features purified, isolated, orrecombinant polynucleotides encoding said KRIB polypeptides described inthe first aspect, or the complement thereof. In further embodiments thepolynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, anddouble-stranded.

In a third aspect, the invention features a recombinant vectorcomprising, consisting essentially of, or consisting of, saidpolynucleotide described in the second aspect.

In a fourth aspect, the invention features a recombinant cellcomprising, consisting essentially of, or consisting of said recombinantvector described in the third aspect. A further embodiment includes ahost cell recombinant for a polynucleotide of the invention.

In a fifth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said KRIB polypeptides described inthe first aspect and, alternatively, a pharmaceutical or physiologicallyacceptable diluent.

In a sixth aspect, the invention features a method of reducing body masscomprising providing or administering to individuals in need of reducingbody mass said pharmaceutical or physiologically acceptable compositiondescribed in the fifth aspect.

In a further preferred embodiment, the invention features a method ofcomplementary therapy of reducing body mass comprising providing oradministering to individuals in need of reducing body mass saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect in combination with a weight reducing agent. Examplesof said weight reducing agent include lipase inhibitors, such asorlistat, and serotonin reuptake inhibitors (SSRI) and noradrenalinereuptake inhibitor, such as sibutramine.

In further preferred embodiments, the invention features a method ofmaintaining a reduced body mass comprising providing or administering toindividuals in need of maintaining a reduced body mass saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect. Further preferred is a method of maintaining a reducedbody fat mass that comprises providing or administering to individualsin need thereof said pharmaceutical or physiologically acceptablecomposition described in the fifth aspect, returning energy intake to anormal level in said individual, and maintaining increased energyexpenditure in said individual. Preferably, said individual is able tomaintain a stable weight that is 10-20% below said individual's obeseweight (as defined herein). In preferred embodiments, said individual isa mammal, preferably a human.

In other preferred embodiments, the invention features a method ofmaintaining weight loss comprising providing or administering toindividuals in need of maintaining weight loss said pharmaceutical orphysiologically acceptable composition described in the fifth aspect incombination with reduced energy intake and/or increased energyexpenditure.

In preferred embodiments, the identification of said individuals in needof reducing body mass to be treated with said pharmaceutical orphysiologically acceptable composition comprises genotyping KRIB singlenucleotide polymorphisms (SNPs) or measuring KRIB polypeptide or mRNAlevels in clinical samples from said individuals. Preferably, saidclinical samples are selected from the group consisting of plasma,urine, and saliva. Preferably, a KRIB polypeptide fragment of thepresent invention is administered to an individual with at least a 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood,serum or plasma levels of full-length any one or all of the KRIBpolypeptides or the naturally proteolytically cleaved KRIB fragments ascompared to healthy, non-obese patients.

In a seventh aspect, the invention features a method of preventing ortreating a metabolic disorder comprising providing or administering toan individual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the fifth aspect. Inpreferred embodiments, the identification of said individuals in need ofsuch treatment to be treated with said pharmaceutical or physiologicallyacceptable composition comprises genotyping KRIB single nucleotidepolymorphisms (SNPs) or measuring KRIB polypeptide or mRNA levels inclinical samples from said individuals. Preferably, said clinicalsamples are selected from the group consisting of blood, serum, plasma,urine, and saliva. Preferably, said metabolic disorder is selected fromthe group consisting of obesity, impaired glucose tolerance, insulinresistance, atherosclerosis, atheromatous disorder, heart disorder,hypertension, stroke, Syndrome X, non-insulin-dependent diabetes andType II diabetes. Type II diabetes-related complications to be treatedby the methods of the invention include microangiopathic lesions, ocularlesions, and renal lesions. Heart disorder includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other metabolic disorders to be treated by compounds of theinvention include hyperlipidemia and hyperuricemia. Yet other metabolicdisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia. Inpreferred embodiments, said individual is a mammal, preferably a human.

In related aspects, embodiments of the present invention includesmethods of causing or inducing a desired biological response in anindividual comprising the steps of: providing or administering to anindividual a composition comprising a KRIB polypeptide, wherein saidbiological response is selected from the group consisting of:

-   -   (a) modulating circulating (either blood, serum, or plasma)        levels (concentration) of free fatty acids, wherein said        modulating is preferably lowering;    -   (b) modulating circulating (either blood, serum or plasma)        levels (concentration) of glucose, wherein said modulating is        preferably lowering;    -   (c) modulating circulating (either blood, serum or plasma)        levels (concentration) of triglycerides, wherein said modulating        is preferably lowering;    -   (d) stimulating muscle lipid or free fatty acid oxidation;    -   (c) modulating leptin uptake in the liver or liver cells,        wherein said modulating is preferably increasing;    -   (e) modulating the postprandial increase in plasma free fatty        acids due to a high fat meal, wherein said modulating is        preferably reducing;    -   (f) modulating ketone body production as the result of a high        fat meal, wherein said modulating is preferably reducing or        eliminating;    -   (g) increasing cell or tissue sensitivity to insulin,        particularly muscle, adipose, liver or brain; and    -   (h) inhibiting the progression from impaired glucose tolerance        to insulin resistance;    -   and further wherein said biological response is significantly        greater than, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100%        or 500% greater than, the biological response caused or induced        by insulin alone at the same molar concentration. In further        preferred embodiments, the present invention of said        pharmaceutical or physiologically acceptable composition can be        used as a method to control blood glucose in some persons with        Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II        diabetes) in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy of NIDDM patients to improve their weight orglucose control in combination with an insulin secretagogue (preferablyoral form) or an insulin sensitising (preferably oral form) agent.Preferably, the oral insulin secretagogue is1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of NIDDM patients alone, without an insulinsecretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may be used incomplementary therapy of NIDDM patients to improve their weight orglucose control in combination with an insulin secretagogue (preferablyoral form) or an insulin sensitising (preferably oral form) agent.Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected fromtolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride,glipizide and glidazide. Preferably, the insulin sensitising agent isselected from metformin, ciglitazone, troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of IDDM patients alone, without an insulinsecretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin NIDDM or IDDM patients.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with InsulinDependent Diabetes Mellitus (IDDM, Type I diabetes) in combination withinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulintherapy.

In an eighth aspect, the invention features a method of making the KRIBpolypeptides described in the first aspect, wherein said method isselected from the group consisting of: proteolytic cleavage, recombinantmethodology and artificial synthesis.

In a ninth aspect, the present invention provides a method of making arecombinant KRIB polypeptide fragment or a full length KRIB polypeptide,the method comprising providing a transgenic, non-human mammal whosemilk contains said recombinant KRIB polypeptide fragment or full-lengthprotein, and purifying said recombinant KRIB polypeptide fragment orsaid full-length KRIB polypeptide from the milk of said non-humanmammal. In one embodiment, said non-human mammal is a cow, goat, sheep,rabbit, or mouse. In another embodiment, the method comprises purifyingrecombinant mature KRIB polypeptide absent the signal peptide from saidmilk, and further comprises cleaving said protein in vitro to obtain adesired KRIB polypeptide fragment.

In a tenth aspect, the invention features a use of the polypeptidedescribed in the first aspect for the preparation of a medicament forthe treatment of obesity-related disorders and/or for reducing bodymass. Preferably, said metabolic disorders are selected from the groupconsisting of obesity, insulin resistance, atherosclerosis, atheromatousdisorder, heart disorder, hypertension, stroke, Syndrome X,non-insulin-dependent diabetes and Type II diabetes. Type IIdiabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, and renallesions. Heart disorder includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic disorders to be treated by compounds of the invention includehyperlipidemia and hyperuricemia. Yet other metabolic disorders of theinvention include cachexia, wasting, AIDS-related weight loss, anorexia,and bulimia. In preferred embodiments, said individual is a mammal,preferably a human.

The invention further features a use of the polypeptide described in thefirst aspect for the preparation of a medicament for prevention ofweight gain, for weight reduction, and/or for maintenance of weightloss. In preferred embodiments, said individual is a mammal, preferablya human.

In an eleventh aspect, the invention features a use of the polypeptidedescribed in the first aspect for treatment of metabolic disordersand/or reducing or increasing body mass. Preferably, said metabolicdisorders are selected from the group consisting of obesity, insulinresistance, atherosclerosis, atheromatous disorder, heart disorder,hypertension, stroke, Syndrome X, non-insulin-dependent diabetes andType II diabetes. Type II diabetes-related complications to be treatedby the methods of the invention include microangiopathic lesions, ocularlesions, and renal lesions. Heart disorder includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other metabolic disorders to be treated by compounds of theinvention include hyperlipidemia and hyperuricemia. Yet other metabolicdisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, anorexia, and bulimia. In preferred embodiments, saidindividual is a mammal, preferably a human.

The invention further features a use of the polypeptide described in thefirst aspect for prevention of weight gain, for weight reduction, and/orfor maintenance of weight loss. In preferred embodiments, saidindividual is a mammal, preferably a human.

In a twelfth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a composition of the fifth aspect of theinvention, for use in a method of treatment of the human or animal body.

In a thirteenth aspect, the invention features methods of reducing bodyweight for cosmetic purposes comprising providing to an individual saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect, or a polypeptide described in the first aspect.Preferably, for said reducing body weight said individual has a BMI ofat least 20 and no more than 25. Alternatively, for said increasing bodyweight said individual preferably has a BMI of at least 15 and no morethan 20.

In a related aspect, the invention features methods of maintainingweight loss comprising providing to an individual said pharmaceutical orphysiologically acceptable composition described in the fifth aspect, orthe polypeptide described in the first aspect. Where the maintenance ofweight loss is practiced for cosmetic purposes, the individual has a BMIof at least 20 and no more than 25. In embodiments for the treatment ofobesity by means of maintaining weight loss, the individual may have aBMI of at least 20. One embodiment for the treatment of obesity by meansof maintaining weight loss provides for the treatment of individualswith BMI values of at least 25. Another embodiment for the treatment ofobesity by means of maintaining weight loss provides for the treatmentof individuals with BMI values of at least 30.

In a fourteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body mass and/or for treatment or prevention of metabolicdisorders. Preferably, said metabolic disorder is selected from thegroup consisting of obesity, impaired glucose tolerance, insulinresistance, atherosclerosis, atheromatous disorder, heart disorder,hypertension, stroke, Syndrome X, non-insulin-dependent diabetes andType II diabetes. Type II diabetes-related complications to be treatedby the methods of the invention include microangiopathic lesions, ocularlesions, and renal lesions. Heart disorder includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other metabolic disorders to be treated by compounds of theinvention include hyperlipidemia and hyperuricemia. Yet other metabolicdisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia. Inpreferred embodiments, said individual is a mammal, preferably a human.In preferred embodiments, the identification of said individuals to betreated with said pharmaceutical or physiologically acceptablecomposition comprises genotyping KRIB single nucleotide polymorphisms(SNPs) or measuring KRIB polypeptides or mRNA levels in clinical samplesfrom said individuals. Preferably, said clinical samples are selectedfrom the group consisting of blood, serum, plasma, urine, and saliva.

In a fifteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body weight for cosmetic reasons.

In a related aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect formaintaining weight loss for cosmetic reasons.

In a sixteenth aspect, the invention features methods of treatinginsulin resistance comprising providing to an individual saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect, or a polypeptide described in the first aspect.

In a seventeenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect ina method of treating individuals with normal glucose tolerance (NGT) whoare obese or who have fasting hyperinsulinemia, or who have both.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with gestationaldiabetes. Gestational diabetes refers to the development of diabetes inan individual during pregnancy, usually during the second or thirdtrimester of pregnancy.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with impairedfasting glucose (IFG). Impaired fasting glucose (IFG) is that conditionin which fasting plasma glucose levels in an individual are elevated butnot diagnostic of overt diabetes, i.e. plasma glucose levels of lessthan 126 mg/dl and less than or equal to 110 mg/dl.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating and preventing impaired glucosetolerance (IGT) in an individual. By providing therapeutics and methodsfor reducing or preventing IGT, i.e., for normalizing insulinresistance, the progression to NIDDM can be delayed or prevented.Furthermore, by providing therapeutics and methods for reducing orpreventing insulin resistance, the invention provides methods forreducing and/or preventing the appearance of Insulin-ResistanceSyndrome.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having polycysticovary syndrome (PCOS). PCOS is among the most common disorders ofpremenopausal women, affecting 5-10% of this population.Insulin-sensitizing agents, e.g., troglitazone, have been shown to beeffective in PCOS and that, in particular, the defects in insulinaction, insulin secretion, ovarian steroidogenosis and fibrinolysis areimproved (Ehrman et al. (1997) J Clin Invest 100:1230), such as ininsulin-resistant humans. Accordingly, the invention provides methodsfor reducing insulin resistance, normalizing blood glucose thus treatingand/or preventing PCOS.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having insulinresistance.

In further preferred embodiments, a subject having insulin resistance istreated according to the methods of the invention to reduce or cure theinsulin-resistance. As insulin resistance is also often associated withinfections and cancer, prevention or reducing insulin resistanceaccording to the methods of the invention may prevent or reduceinfections and cancer.

In further preferred embodiment, the methods of the invention are usedto prevent the development of insulin resistance in a subject, e.g.,those known to have an increased risk of developing insulin-resistance.

Thus, any of the above-described tests or other tests known in the artcan be used to determine that a subject is insulin-resistant, whichpatient can then be treated according to the methods of the invention toreduce or cure the insulin-resistance. Alternatively, the methods of theinvention can also be used to prevent the development of insulinresistance in a subject, e.g., those known to have an increased risk ofdeveloping insulin-resistance.

In an eighteenth aspect, the invention features a method of preventingor treating an metabolic disorder comprising providing or administeringto an individual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the fifth aspect. Inpreferred embodiments, the identification of said individuals in need ofsuch treatment to be treated with said pharmaceutical or physiologicallyacceptable composition comprises genotyping KRIB single nucleotidepolymorphisms (SNPs) or measuring KRIB polypeptide or mRNA levels inclinical samples from said individuals. Preferably, said clinicalsamples are selected from the group consisting of blood, serum, plasma,urine, and saliva. Preferably, said metabolic disorder is selected fromthe group consisting of obesity, impaired glucose tolerance, insulinresistance, atherosclerosis, atheromatous disorder, heart disorder,hypertension, stroke, Syndrome X, non-insulin-dependent diabetes andType II diabetes. Type II diabetes-related complications to be treatedby the methods of the invention include microangiopathic lesions, ocularlesions, and renal lesions. Heart disorder includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other metabolic disorders to be treated by compounds of theinvention include hyperlipidemia and hyperuricemia. Yet other metabolicdisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia. Inpreferred embodiments, said individual is a mammal, preferably a human.

In a nineteenth aspect, the invention features a method of using a KRIBpolypeptide to screen compounds for one or more antagonists of KRIBpolypeptide activity, wherein said activity is selected from but notrestricted to lipid partitioning, lipid metabolism, and insulin-likeactivity.

In a related aspect, the invention features a method of using a KRIBpolypeptide to screen compounds for one or more antagonists of KRIBpolypeptide activity, wherein said activity is selected from but notrestricted to prevention of weight gain, weight reduction, andmaintenance of weight loss.

In preferred embodiment, said compound is selected from but is notrestricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid.

In a twentieth aspect, the invention features a method of using a KRIBpolypeptide to identify one or more cell types expressing a cell surfacereceptor for said KRIB polypeptide, preferably wherein said polypeptidehas lipid partitioning, lipid metabolism, or insulin-like activities.

In a twenty-first aspect, the invention features a method of using aKRIB polypeptide to clone cDNA encoding a cell surface receptor for saidKRIB polypeptide, preferably wherein said polypeptide has lipidpartitioning, lipid metabolism, or insulin-like activities.

In a twenty-second aspect, the invention features a method of using aKRIB polynucleotide to generate transgenic non-human mammals expressingKRIB polypeptides, preferably wherein said non-human mammal is mouse,cow, sheep, goat, pig, or rabbit.

In a twenty-third aspect, the invention features a purified or isolatedantibody capable of specifically binding to a polypeptide of the presentinvention. In one aspect of this embodiment, the antibody is capable ofbinding to a polypeptide comprising at least 6 consecutive amino acids,at least 8 consecutive amino acids, or at least 10 consecutive aminoacids of even SEQ ID NOs: 2-10.

In a preferred aspect of the methods above and disclosed herein, theamount of KRIB polypeptide or polynucleotide administered to anindividual is sufficient to bring circulating (blood, serum, or plasma)levels (concentration) of KRIB polypeptides to their normal levels(levels in non-obese individuals). “Normal levels” may be specified asthe total concentration of all circulating KRIB polypeptides(full-length KRIB proteins and fragments thereof) or the concentrationof all circulating proteolytically cleaved KRIB polypeptides only.

In a further preferred aspect of the methods above and disclosed herein,weight loss is due in part or in whole to a decrease in mass of eithera) subcutaneous adipose tissue and/or b) visceral (omental) adiposetissue.

A highly preferred embodiment of the present invention is directed tothe use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,Syndrome X, and Type II diabetes, wherein said KRIB polypeptide isselected from the group consisting of: (a) a KRIB-1 polypeptidecomprising at least 6 amino acids of SEQ ID NO: 2; (b) a KRIB-2polypeptide comprising at least 6 amino acids of SEQ ID NO:4; (c) aKRIB-2R polypeptide comprising at least 6 amino acids of SEQ ID NO: 6;(d) a KRIB-3 polypeptide comprising at least 6 amino acids of SEQ ID NO:8; and (e) a KRIB-4 polypeptide comprising at least 6 amino acids of SEQID NO: 10.

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for screening for KRIB ligands, whereinsaid KRIB polypeptide is selected from the group consisting of: (a) aKRIB-1 polypeptide comprising at least 6 amino acids of SEQ ID NO: 2;(b) a KRIB-2 polypeptide comprising at least 6 amino acids of SEQ IDNO:4; (c) a KRIB-2R polypeptide comprising at least 6 amino acids of SEQID NO: 6; (d) a KRIB-3 polypeptide comprising at least 6 amino acids ofSEQ ID NO: 8; and (e) a KRIB-4 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 10.

Another highly preferred embodiment of the present invention is directedto the use of a KRIB ligand for screening for KRIB ligands, wherein theKRIB ligand is a KRIB antagonist or a KRIB agonist.

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder, or for screening for KRIBligands, wherein said KRIB polypeptide is a KRIB-1 polypeptide selectedfrom the group consisting of: (a) a polypeptide comprising SEQ ID NO: 2;(b) a polypeptide comprising amino acids 20 to 136 of SEQ ID NO: 2; (c)a variant of (a) or (b), wherein the amino acid sequence has at least50% or 60% or 70% or 80% or 90% identity to at least one of thesequences in (a) or (b); (d) a variant of (a) or (b) which is encoded bya DNA sequence which hybridizes to the complement of the DNA sequenceencoding (a) or (b) under moderately stringent conditions or underhighly stringent conditions; and (e) a variant of (a) or (b) wherein anychanges in the amino acid sequence are conservative amino acidsubstitutions to the amino acid sequences in (a) or (b).

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder, or for screening for KRIBligands, wherein said KRIB polypeptide is a KRIB-2 polypeptide selectedfrom the group consisting of: (a) a polypeptide comprising SEQ ID NO: 4;(b) a polypeptide comprising amino acids 15 to 97 of SEQ ID NO: 4; (c) apolypeptide comprising the LAWN Domain region at amino acids positions72 to 94 of SEQ ID NO: 4; (d) a variant of any of (a) to (d), whereinthe amino acid sequence has at least 50% or 60% or 70% or 80% or 90%identity to at least one of the sequences in (a) to (d); (e) a variantof any of (a) to (d) which is encoded by a DNA sequence which hybridizesto the complement of the DNA sequence encoding any of (a) to (d) undermoderately stringent conditions or under highly stringent conditions;and (f) a variant of any of (a) to (d) wherein any changes in the aminoacid sequence are conservative amino acid substitutions to the aminoacid sequences in (a) to (d).

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder, or for screening for KRIBligands, wherein said KRIB polypeptide is a KRIB-2R polypeptide selectedfrom the group consisting of: (a) a polypeptide comprising SEQ ID NO: 6;(b) a polypeptide comprising amino acids 15 to 111 of SEQ ID NO: 6; (c)A polypeptide comprising the LAWN Domain region at amino acids positions39 to 61 of SEQ ID NO: 6. (d) a variant of any of (a) to (c), whereinthe amino acid sequence has at least 50% or 60% or 70% or 80% or 90%identity to at least one of the sequences in (a) to (c); (e) a variantof any of (a) to (c) which is encoded by a DNA sequence which hybridizesto the complement of the DNA sequence encoding any of (a) to (c) undermoderately stringent conditions or under highly stringent conditions;and (f) a variant of any of (a) to (c) wherein any changes in the aminoacid sequence are conservative amino acid substitutions to the aminoacid sequences in (a) to (c).

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder, or for screening for KRIBligands, wherein said KRIB polypeptide is a KRIB-3 polypeptide selectedfrom the group consisting of: (a) a polypeptide comprising SEQ ID NO: 8;(b) a polypeptide comprising amino acids 18 to 212 of SEQ ID NO: 8; (c)a variant of (a) or (b), wherein the amino acid sequence has at least50% or 60% or 70% or 80% or 90% identity to at least one of thesequences in (a) or (b); (d) a variant of (a) or (b) which is encoded bya DNA sequence which hybridizes to the complement of the DNA sequenceencoding (a) or (b) under moderately stringent conditions or underhighly stringent conditions; and (e) a variant of (a) or (b) wherein anychanges in the amino acid sequence are conservative amino acidsubstitutions to the amino acid sequences in (a) or (b).

Another highly preferred embodiment of the present invention is directedto the use of a KRIB polypeptide for the preparation of a medicament fortreating or preventing a metabolic disorder, or for screening for KRIBligands, wherein said KRIB polypeptide is a KRIB-4 polypeptidepolypeptide selected from the group consisting of: (a) a polypeptidecomprising SEQ ID NO: 10; (b) a polypeptide comprising amino acids 36 to347 of SEQ ID NO: 10; (c) a polypeptide comprising one of the leucinerich repeat domains at amino acid positions 93 to 116, 117 to 140, 141to 164, 165 to 188, 189 to 212, 213 to 236, 237 to 260 and 261 to 284 ofSEQ ID NO: 10; (d) a polypeptide comprising one of the segmentsexhibiting a periodic pattern in the occurrence of leucine, proline, andasparagines at amino acid positions 84 to 107, 109 to 131, 132 to 155,156 to 179, 180 to 203, 204 to 227, 228 to 251 and 252 to 275 of SEQ IDNO: 10; (e) a polypeptide comprising the leucine rich repeat C-terminaldomain at amino acid positions 299 to 347 of SEQ ID NO: 10; (f) aAvariant of any of (a) to (e), wherein the amino acid sequence has atleast 50% or 60% or 70% or 80% or 90% identity to at least one of thesequences in (a) to (e); (g) a variant of any of (a) to (e) which isencoded by a DNA sequence which hybridizes to the complement of the DNAsequence encoding any of (a) to (e) under moderately stringentconditions or under highly stringent conditions; and (h) a variant ofany of (a) to (e) wherein any changes in the amino acid sequence areconservative amino acid substitutions to the amino acid sequences in (a)to (e).

Another highly preferred embodiment of the present invention is directedto the use of a KRIB agonist for the preparation of a medicament fortreating or preventing a metabolic disorder selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,Syndrome X, and Type II diabetes.

Another highly preferred embodiment of the present invention is directedto the use of a KRIB antagonist for the preparation of a medicament fortreating or preventing a metabolic disorder selected from the groupconsisting of cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, anorexia, and bulimia.

Another highly preferred embodiment of the present invention is directedto the use of a KRIB antagonist for the preparation of a medicament fortreating or preventing a metabolic disorder, wherein said KRIBantagonist is an antibody.

Another highly preferred embodiment of the present invention is directedto the use of a gene therapy vector comprising a nucleotide sequenceencoding a KRIB polypeptide for the preparation of a medicament fortreating and preventing a metabolic disorder selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,Syndrome X, and Type II diabetes, wherein said KRIB is selected from thegroup consisting of: (a) a KRIB-1 polypeptide comprising SEQ ID NO: 2;(b) a KRIB-2 polypeptide comprising SEQ ID NO:4; (c) a KRIB-2Rpolypeptide comprising SEQ ID NO: 6; (d) a KRIB-3 polypeptide comprisingSEQ ID NO: 8; and (e) a KRIB-4 polypeptide comprising SEQ ID NO: 10.

Another highly preferred embodiment of the present invention is directedto a method of identifying a KRIB ligand, said method comprising thesteps of: (a) contacting said KRIB polypeptide with a test compound;wherein said KRIB polypeptide is selected from the group consisting of:(i) a KRIB-1 polypeptide comprising at least 6 amino acids of SEQ ID NO:2; (ii) a KRIB-2 polypeptide comprising at least 6 amino acids of SEQ IDNO:4; (iii) a KRIB-2R polypeptide comprising at least 6 amino acids ofSEQ ID NO: 6; (iv) a KRIB-3 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 8; and (v) a KRIB-4 polypeptide comprising at least6 amino acids of SEQ ID NO: 10; and (b) determining whether saidcompound specifically binds to said polypeptide, wherein a detectionthat said compound specifically binds to said polypeptide indicates thatsaid compound is a ligand of said KRIB polypeptide.

Another highly preferred embodiment of the present invention is directedto a method of screening candidate compounds for a KRIB agonist or aKRIB antagonist comprising the steps of: (a) contacting a cell with aKRIB polypeptide with or without a candidate compound, wherein said KRIBpolypeptide is selected from the group consisting of: (i) a KRIB-1polypeptide comprising at least 6 amino acids of SEQ ID NO: 2; (ii) aKRIB-2 polypeptide or comprising at least 6 amino acids of SEQ ID NO:4;(iii) a KRIB-2R polypeptide comprising at least 6 amino acids of or SEQID NO: 6; (iv) a KRIB-3 polypeptide comprising at least 6 amino acids ofSEQ ID NO: 8; and (v) a KRIB-4 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 10; and (b) detecting a result on the basis ofactivity, wherein said activity is selected from the group consistingof: LSR expression, leptin transport, increase in glucose uptake,decrease in blood lipid or triglyceride levels, increase in lipoproteinbinding, uptake or degradation, and increase in free fatty acidoxidation; wherein said result identifies said candidate compound as anagonists or antagonist of a KRIB polypeptide activity if said resultwith compound differs from said result without compound.

Another highly preferred embodiment of the present invention is directedto a method of screening candidate compounds for a KRIB agonist or aKRIB antagonist comprising the steps of (a) administering to a testanimal a KRIB polypeptide with or without a candidate compound, whereinsaid KRIB polypeptide is selected from the group consisting of: (i) aKRIB-1 polypeptide comprising at least 6 amino acids of SEQ ID NO: 2;(ii) a KRIB-2 polypeptide or comprising at least 6 amino acids of SEQ IDNO:4; (iii) a KRIB-2R polypeptide comprising at least 6 amino acids ofor SEQ ID NO: 6; (iv) a KRIB-3 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 8; and (v) a KRIB-4 polypeptide comprising at least6 amino acids of SEQ ID NO: 10; and (b) detecting a result on the basisof activity, wherein said activity is selected from the group consistingof: prevention of weight gain, weight reduction, and maintenance ofweight loss; wherein said result identifies said candidate compound asan agonists or antagonist of a KRIB polypeptide activity if said resultwith compound differs from said result without compound.

DETAILED DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 represents the cDNA sequence of KRIB-1.

SEQ ID NO: 2 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO: 1.

SEQ ID NO: 3 represents the cDNA sequence of KRIB-2.

SEQ ID NO: 4 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO: 3.

SEQ ID NO: 5 represents the cDNA sequence of KRIB-2R.

SEQ ID NO: 6 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO: 5.

SEQ ID NO: 7 represents the cDNA sequence of KRIB-3.

SEQ ID NO: 8 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO: 7.

SEQ ID NO: 9 represents the cDNA sequence of KRIB-4.

SEQ ID NO: 10 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO: 9.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

As used interchangeably herein, the terms “oligonucleotides”, and“polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybridsequences of more than one nucleotide in either single chain or duplexform. The terms encompass “modified nucleotides” which comprise at leastone modification, including by way of example and not limitation: (a) analternative linking group, (b) an analogous form of purine, (c) ananalogous form of pyrimidine, or (d) an analogous sugar. For examples ofanalogous linking groups, purines, pyrimidines, and sugars see forexample PCT publication No. WO 95/04064. The polynucleotide sequences ofthe invention may be prepared by any known method, including synthetic,recombinant, ex vivo generation, or a combination thereof, as well asutilizing any purification methods known in the art.

The terms polynucleotide construct, recombinant polynucleotide andrecombinant polypeptide are used herein consistently with their use inthe art. The terms “upstream” and “downstream” are also used hereinconsistently with their use in the art. The terms “base paired” and“Watson & Crick base paired” are used interchangeably herein andconsistently with their use in the art. Similarly, the terms“complementary”, “complement thereof”, “complement”, “complementarypolynucleotide”, “complementary nucleic acid” and “complementarynucleotide sequence” are used interchangeably herein and consistentlywith their use in the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention that has been separated fromother compounds including, but not limited to, other nucleic acids,carbohydrates, lipids and proteins (such as the enzymes used in thesynthesis of the polynucleotide). Purified can also refer to theseparation of covalently closed polynucleotides from linearpolynucleotides, or vice versa, for example. A polynucleotide issubstantially pure when at least about 50%, 60%, 75%, or 90% of a samplecontains a single polynucleotide sequence. In some cases this involves adetermination between conformations (linear versus covalently closed). Asubstantially pure polynucleotide typically comprises about 50, 60, 70,80, 90, 95, 99% weight/weight of a nucleic acid sample. Polynucleotidepurity or homogeneity may be indicated by a number of means well knownin the art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes, higher resolution can beachieved by using HPLC or other means well known in the art.

Similarly, the term “purified” is used herein to describe a polypeptideof the invention that has been separated from other compounds including,but not limited to, nucleic acids, lipids, carbohydrates and otherproteins. In some preferred embodiments, a polypeptide is substantiallypure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.5% of the polypeptide molecules of a sample have a singleamino acid sequence. In some preferred embodiments, a substantially purepolypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%,97%, 98%, 99% or 99.5% weight/weight of a protein sample. Polypeptidepurity or homogeneity is indicated by a number of methods well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polypeptide band upon stainingthe gel. For certain purposes, higher resolution can be achieved byusing HPLC or other methods well known in the art.

Further, as used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition. Purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated. Alternatively,purification may be expressed as “at least” a percent purity relative toheterologous polynucleotides (DNA, RNA or both) or polypeptides. As apreferred embodiment, the polynucleotides or polypeptides of the presentinvention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologouspolynucleotides or polypeptides. As a further preferred embodiment thepolynucleotides or polypeptides have an “at least” purity ranging fromany number, to the thousandth position, between 90% and 100% (e.g., atleast 99.995% pure) relative to heterologous polynucleotides orpolypeptides. Additionally, purity of the polynucleotides orpolypeptides may be expressed as a percentage (as described above)relative to all materials and compounds other than the carrier solution.Each number, to the thousandth position, may be claimed as individualspecies of purity.

The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

Specifically excluded from the definition of “isolated” are: naturallyoccurring chromosomes (e.g., chromosome spreads), artificial chromosomelibraries, genomic libraries, and cDNA libraries that exist either as anin vitro nucleic acid preparation or as a transfected/transformed hostcell preparation, wherein the host cells are either an in vitroheterogeneous preparation or plated as a heterogeneous population ofsingle colonies. Also specifically excluded are the above librarieswherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%,10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acidinserts in the vector molecules. Further specifically excluded are wholecell genomic DNA or whole cell RNA preparations (including said wholecell preparations which are mechanically sheared or enzymaticallydigested). Further specifically excluded are the above whole cellpreparations as either an in vitro preparation or as a heterogeneousmixture separated by electrophoresis (including blot transfers of thesame) wherein the polynucleotide of the invention have not been furtherseparated from the heterologous polynucleotides in the electrophoresismedium (e.g., further separating by excising a single band from aheterogeneous band population in an agarose gel or nylon blot).

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by DNA polymerase, RNApolymerase, or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment that can be usedto identify a specific polynucleotide sequence present in a sample, saidnucleic acid segment comprising a nucleotide sequence complementary tothe specific polynucleotide sequence to be identified.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer. Thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides. For example, polypeptides that include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

Without being limited by theory, the compounds/polypeptides of theinvention are capable of modulating the partitioning of dietary lipidsbetween the liver and peripheral tissues, and thus of treating“disorders involving the partitioning of dietary lipids between theliver and peripheral tissues.” The term “peripheral tissues” is meant toinclude muscle and adipose tissue. In preferred embodiments, thecompounds/polypeptides of the invention partition the dietary lipidstoward the muscle. In alternative preferred embodiments, the dietarylipids are partitioned toward the adipose tissue. In other preferredembodiments, the dietary lipids are partitioned toward the liver. In yetother preferred embodiments, the compounds/polypeptides of the inventionincrease or decrease the oxidation of dietary lipids, preferably freefatty acids (FFA) by the muscle. Dietary lipids include, but are notlimited to triglycerides and free fatty acids.

Preferred disorders believed to involve the partitioning of dietarylipids include obesity and obesity-related disorders such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disorder, heart disorder, hypertension, stroke, Syndrome X,Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions. Heart disorder includes, but is notlimited to, cardiac insufficiency, coronary insufficiency, and highblood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related disorders of the invention include cachexia,wasting, AIDS-related weight loss, cancer-related weight loss, anorexia,and bulimia.

The term “heterologous”, when used herein, is intended to designate anypolypeptide or polynucleotide other than a KRIB polypeptide or apolynucleotide encoding a KRIB polypeptide of the present invention.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. A defined meaning setforth in the M. P.E.P. controls over a defined meaning in the art and adefined meaning set forth in controlling Federal Circuit case lawcontrols over a meaning set forth in the M.P.E.P. With this in mind, theterms may be substituted for one another throughout the instantapplication in order to attach the specific meaning associated with eachterm.

The term “host cell recombinant for” a particular polynucleotide of thepresent invention, means a host cell that has been altered by the handsof man to contain said polynucleotide in a way not naturally found insaid cell. For example, said host cell may be transiently or stablytransfected or transduced with said polynucleotide of the presentinvention.

The term “obesity” as used herein is defined in the WHO classificationsof weight (Kopelman (2000) Nature 404:635643). Underweight is less than18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade3 overweight is greater than or equal to 40.0 BMI. BMI is body massindex (morbid obesity) and is kg/m². Waist circumference can also beused to indicate a risk of metabolic complications where in men acircumference of greater than or equal to 94 cm indicates an increasedrisk, and greater than or equal to 102 cm indicates a substantiallyincreased risk. Similarly for women, greater than or equal to 88 cmindicates an increased risk, and greater than or equal to 88 cmindicates a substantially increased risk. The waist circumference ismeasured in cm at midpoint between lower border of ribs and upper borderof the pelvis. Other measures of obesity include, but are not limitedto, skinfold thickness which is a measurement in cm of skinfoldthickness using calipers, and bioimpedance, which is based on theprinciple that lean mass conducts current better than fat mass becauseit is primarily an electrolyte solution; measurement of resistance to aweak current (impedance) applied across extremities provides an estimateof body fat using an empirically derived equation.

The term “energy intake” as used herein is defined as the energyintroduced into an individual from total caloric intake, i.e., the totalenergy from food and liquid diet. The term “energy expenditure” as usedherein is defined as total energy expenditure (TEE), which includesresting energy expenditure (REE), the thermic effect of feeding (TEF),and activities such as exercise. Both “energy intake” and “energyexpenditure” are further defined by Rosenbaum et al. [Am J Clin Nutr(2000) June; 71(6): 1421-32), which is hereby incorporated by referencein its entirety].

The term “maintenance of weight loss” as used herein is defined assustaining a stable weight in an individual that is 10-20% below theinitial, obese weight of the individual. Preferably, the new maintainedweight after weight loss is a healthy weight (as defined herein). Whenthe maintenance of weight loss is practiced for cosmetic purposes, theindividual has a BMI of at least 20 and no more than 25. As defined forthe treatment of obesity by means of maintaining weight loss, theindividual may have a BMI of at least 20.

The term “diabetes” as used herein is intended to encompass the usualdiagnosis of diabetes made from any of the methods included, but notlimited to, the following list: symptoms of diabetes (eg. polyuria,polydipsia, polyphagia) plus casual plasma glucose levels of greaterthan or equal to 200 mg/dl, wherein casual plasma glucose is defined anytime of the day regardless of the timing of meal or drink consumption; 8hour fasting plasma glucose levels of less than or equal to 126 mg/dl;and plasma glucose levels of greater than or equal to 200 mg/dl 2 hoursfollowing oral administration of 75 g anhydrous glucose dissolved inwater.

The term “impaired glucose tolerance (IGT)” as used herein is intendedto indicate that condition associated with insulin-resistance that isintermediate between frank, NIDDM and normal glucose tolerance (NGT). Ahigh percentage of the IGT population is known to progress to NIDDMrelative to persons with normal glucose tolerance (Sad et al., New EnglJ Med 1988; 319:1500-6). Thus, by providing therapeutics and methods forreducing or preventing IGT, i.e., for normalizing insulin resistance,the progression to NIDDM can be delayed or prevented. IGT is diagnosedby a procedure wherein an affected person's postprandial glucoseresponse is determined to be abnormal as assessed by 2-hour postprandialplasma glucose levels. In this test, a measured amount of glucose isgiven to the patient and blood glucose levels measured regularintervals, usually every half hour for the first two hours and everyhour thereafter. In a “normal” or non-IGT individual, glucose levelsrise during the first two hours to a level less than 140 mg/dl and thendrop rapidly. In an IGT individual, the blood glucose levels are higherand the drop-off level is at a slower rate.

The term “Insulin-Resistance Syndrome” as used herein is intended toencompass the cluster of abnormalities resulting from an attempt tocompensate for insulin resistance that sets in motion a series of eventsthat play an important role in the development of both hypertension andcoronary artery disorder (CAD), such as premature atheroscleroticvascular disorder. Increased plasma triglyceride and decreasedHDL-cholesterol concentrations, conditions that are known to beassociated with CAD, have also been reported to be associated withinsulin resistance. Thus, by providing therapeutics and methods forreducing or preventing insulin resistance, the invention providesmethods for reducing and/or preventing the appearance ofinsulin-resistance syndrome.

The term “polycystic ovary syndrome (PCOS)” as used herein is intendedto designate that etiologically unassigned disorder of premenopausalwomen, affecting 5-10% of this population, characterized byhyperandrogenism, chronic anovulation, defects in insulin action,insulin secretion, ovarian steroidogenesis and fibrinolysis. Women withPCOS frequently are insulin resistant and at increased risk to developglucose intolerance or NIDDM in the third and fourth decades of life(Dunaif et al. (1996) J Clin Endocrinol Metab 81:3299). Hyperandrogenismalso is a feature of a variety of diverse insulin-resistant states, fromthe type A syndrome, through leprechaunism and lipoatrophic diabetes, tothe type B syndrome, when these conditions occur in premenopausal women.It has been suggested that hyperinsulinemia per se causeshyperandrogenism. Insulin-sensitizing agents, e.g., troglitazone, havebeen shown to be effective in PCOS and that, in particular, the defectsin insulin action, insulin secretion, ovarian steroidogenosis andfibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100:1230),such as in insulin-resistant humans.

The term “insulin resistance” as used herein is intended to encompassthe usual diagnosis of insulin resistance made by any of a number ofmethods, including but not restricted to: the intravenous glucosetolerance test or measurement of the fasting insulin level. It is wellknown that there is an excellent correlation between the height of thefasting insulin level and the degree of insulin resistance. Therefore,one could use elevated fasting insulin levels as a surrogate marker forinsulin resistance for the purpose of identifying which normal glucosetolerance (NGT) individuals have insulin resistance. Another way to dothis is to follow the approach as disclosed in The New England Journalof Medicine, No. 3, pp. 1188 (1995), i.e. to select obesity as aninitial criterion for entry into the treatment group. Some obesesubjects have impaired glucose tolerance (IGT) while others have normalglucose tolerance (NGT). Since essentially all obese subjects areinsulin resistant, i.e. even the NGT obese subjects are insulinresistant and have fasting hyperinsulinemia. Therefore, the target ofthe treatment according to the present invention can be defined as NGTindividuals who are obese or who have fasting hyperinsulinemia, or whohave both.

A diagnosis of insulin resistance can also be made using the euglycemicglucose clamp test. This test involves the simultaneous administrationof a constant insulin infusion and a variable rate glucose infusion.During the test, which lasts 34 hours, the plasma glucose concentrationis kept constant at euglycemic levels by measuring the glucose levelevery 5-10 minutes and then adjusting the variable rate glucose infusionto keep the plasma glucose level unchanged. Under these circumstances,the rate of glucose entry into the bloodstream is equal to the overallrate of glucose disposal in the body. The difference between the rate ofglucose disposal in the basal state (no insulin infusion) and theinsulin infused state, represents insulin mediated glucose uptake. Innormal individuals, insulin causes brisk and large increase in overallbody glucose disposal, whereas in NIDDM subjects, this effect of insulinis greatly blunted, and is only 20-30% of normal. In insulin resistantsubjects with either IGT or NGT, the rate of insulin stimulated glucosedisposal is about half way between normal and NIDDM. For example, at asteady state plasma insulin concentration of about 100 uU/ml (aphysiologic level) the glucose disposal rate in normal subjects is about7 mg/kg/min. In NIDDM subjects, it is about 2.5 mg/kg/min., and inpatients with IGT (or insulin resistant subjects with NGT) it is about4-5 mg/kg/min. This is a highly reproducible and precise test, and candistinguish patients within these categories. It is also known that assubjects become more insulin resistant, the fasting insulin level rises.There is an excellent positive correlation between the height of thefasting insulin level and the magnitude of the insulin resistance asmeasured by euglycemic glucose clamp tests and, therefore, this providesthe rationale for using fasting insulin levels as a surrogate measure ofinsulin resistance.

The term “agent acting on the partitioning of dietary lipids between theliver and peripheral tissues” refers to a compound or polypeptide of theinvention that modulates the partitioning of dietary lipids between theliver and the peripheral tissues as previously described. Preferably,the agent increases or decreases the oxidation of dietary lipids,preferably free fatty acids (FFA) by the muscle. Preferably the agentdecreases or increases the body weight of individuals or is used totreat or prevent an obesity-related disorder such as obesity, impairedglucose tolerance, insulin resistance, atherosclerosis, atheromatousdisorder, heart disorder, hypertension, stroke, Syndrome X, Non-InsulinDependent Diabetes Mellitus (NIDDM, or Type II diabetes) and InsulinDependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, renallesions. Heart disorder includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other obesity-relateddisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia.

The terms “response to an agent acting on the partitioning of dietarylipids between the liver and peripheral tissues” refer to drug efficacy,including but not limited to, ability to metabolize a compound, abilityto convert a pro-drug to an active drug, and the pharmacokinetics(absorption, distribution, elimination) and the pharmacodynamics(receptor-related) of a drug in an individual.

The terms “side effects to an agent acting on the partitioning ofdietary lipids between the liver and peripheral tissues” refer toadverse effects of therapy resulting from extensions of the principalpharmacological action of the drug or to idiosyncratic adverse reactionsresulting from an interaction of the drug with unique host factors.“Side effects to an agent acting on the partitioning of dietary lipidsbetween the liver and peripheral tissues” can include, but are notlimited to, adverse reactions such as dermatologic, hematologic orhepatologic toxicities and further includes gastric and intestinalulceration, disturbance in platelet function, renal injury, nephritis,vasomotor rhinitis with profuse watery secretions, angioneurotic edema,generalized urticaria, and bronchial asthma to laryngeal edema andbronchoconstriction, hypotension, and shock.

The term “KRIB-related disorders” as used herein refers to any disordercomprising an aberrant functioning of KRIB, or which could be treated orprevented by modulating KRIB levels or activity. “Aberrant functioningof KRIB” includes, but is not limited to, aberrant levels of expressionof KRIB (either increased or decreased, but preferably decreased),aberrant activity of KRIB (either increased or decreased), and aberrantinteractions with ligands or binding partners (either increased ordecreased). By “aberrant” is meant a change from the type, or level ofactivity seen in normal cells, tissues, or patients, or seen previouslyin the cell, tissue, or patient prior to the onset of the illness. Inpreferred embodiments, these KRIB-related disorders include obesity andthe metabolic disorders described previously.

The term “cosmetic treatments” is meant to include treatments withcompounds or polypeptides of the invention that increase or decrease thebody mass of an individual where the individual is not clinically obeseor clinically thin. Thus, these individuals have a body mass index (BMI)below the cut-off for clinical obesity (e.g. below 25 kg/m²) and abovethe cut-off for clinical thinness (e.g. above 18.5 kg/m²). In addition,these individuals are preferably healthy (e.g. do not have an metabolicdisorder of the invention). “Cosmetic treatments” are also meant toencompass, in some circumstances, more localized increases in adiposetissue, for example, gains or losses specifically around the waist orhips, or around the hips and thighs, for example. These localized gainsor losses of adipose tissue can be identified by increases or decreasesin waist or hip size, for example.

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disorder or condition so asto prevent a physical manifestation of aberrations associated withobesity or KRIB.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, etc in thecase of humans; veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

The term “perceives a need for treatment” refers to a sub-clinicaldetermination that an individual desires to reduce weight for cosmeticreasons as discussed under “cosmetic treatment” above. The term“perceives a need for treatment” in other embodiments can refer to thedecision that an owner of an animal makes for cosmetic treatment of theanimal.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

The term “non-human animal” refers to any non-human vertebrate,including birds and more usually mammals, preferably primates, animalssuch as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits orrodents, more preferably rats or mice. Both the terms “animal” and“mammal” expressly embrace human subjects unless preceded with the term“non-human”.

KRIB polypeptides are able to significantly reduce the postprandialresponse of plasma free fatty acids, glucose, and triglycerides inmammals fed a high fat/sucrose meal, while not affecting levels ofleptin, insulin or glucagon. In addition, KRIB polypeptides modulatemuscle free fatty acid oxidation in vitro and ex vivo, preferablyincrease oxidation Further, KRIB polypeptides of the invention modulateweight gain in mammals that are fed a high fat/sucrose diet.

The instant invention encompasses the use of KRIB polypeptides in thepartitioning of free fatty acid (FFA) and as an important new tool tocontrol energy homeostasis. Of the tissues that can significantly removelipids from circulation and cause FFA oxidation, muscle is believed tobe quantitatively the most important.

PREFERRED EMBODIMENTS OF THE INVENTION

I. KRIB Polypeptides of the Invention

KRIB polypeptides have been identified that have measurable activity invitro and in vivo. These activities include, but are not limited to,modulation, preferably reduction, of the postprandial response of plasmafree fatty acids, glucose, and triglycerides in mammals fed a highfat/sucrose meal (Example 6), change, preferably an increase, in musclefree fatty acid oxidation in vitro and ex vivo (Example 10), andsustained weight loss in mammals on a high fat/sucrose diet. Otherassays for KRIB polypeptide activity in vitro and in vivo are alsoprovided (Examples 2, 5, 7, 9, 11, for example), and equivalent assayscan be designed by those with ordinary skill in the art.

KRIB polypeptides are further characterized by elevated expression inhuman adipose tissue (Example 18). KRIB polypeptides are able to lowercirculating (either blood, serum, or plasma) levels (concentration) of:(i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. KRIBpolypeptides are further able to (i) prevent weight gain, (ii) reduceweight, and/or (iii) maintain weight loss.

The term “KRIB polypeptides” includes both the “full-length” polypeptideand fragments of the “full-length” KRIB polypeptides (although each ofthe above species may be particularly specified).

By “intact” or “full-length” KRIB polypeptides as used herein is meantthe full-length polypeptide sequence of any KRIB polypeptide, from theN-terminal methionine to the C-terminal stop codon. Examples of intactor full-length KRIB polypeptides are found in the sequence listing.

The term “metabolic activity” as used herein refers to at least one, andpreferably all, of the activities described herein for KRIBpolypeptides. Assays for the determination of these activities areprovided herein (e.g. Examples 2-14), and equivalent assays can bedesigned by those with ordinary skill in the art. Optionally, “metabolicactivity” can be selected from the group consisting of lipidpartitioning, lipid metabolism, and insulin-like activity, or anactivity within one of these categories. By “lipid partitioning”activity is meant the ability to effect the location of dietary lipidsamong the major tissue groups including, adipose tissue, liver, andmuscle. KRIB polypeptides of the invention play a role in thepartitioning of lipids to the muscle, liver or adipose tissue. By “lipidmetabolism” activity is meant the ability to influence the metabolism oflipids. KRIB polypeptides of the invention have the ability to affectthe level of free fatty acids in the plasma as well as to modulate,preferably increase, the metabolism of lipids in the muscle through freefatty acid oxidation experiments (Examples 2, 6, 8, 9, 10) and totransiently affect the levels of triglycerides in the plasma and themuscle (Examples 6, 8, 11). By “insulin-like” activity is meant theability of KRIB polypeptides to modulate the levels of glucose in theplasma. KRIB polypeptides do not significantly impact insulin levels butdo impact glucose levels similarly to the effects of insulin (Examples 7& 8). These effects may vary in the presence of the intact (full-length)KRIB polypeptides or are significantly greater in the presence of theKRIB polypeptide fragments compared with the full-length KRIBpolypeptides.

The term “significantly greater” as used herein refers to a comparisonof the activity of a KRIB polypeptide in an metabolic assay comparedwith untreated cells in the same assay. By “significantly” as usedherein is meant statistically significant as it is typically determinedby those with ordinary skill in the art. For example, data are typicallycalculated as a mean±SEM, and a p-value ≦0.05 is consideredstatistically significant. Statistical analysis is typically done usingeither the unpaired Student's t test or the paired Student's t test, asappropriate in each study. Examples of a significant change in activityas a result of the presence of a KRIB polypeptide of the inventioncompared to untreated cells include an increase or a decrease in a givenparameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, or 75%. One or more, but not necessarily all, of themeasurable parameters will change significantly in the presence of KRIBpolypeptide as compared to untreated cells.

Representative “metabolic assays” are provided in the Examples. Theseassays include, but are not limited to, methods of measuring thepostprandial response, methods of measuring free fatty acid oxidation,and methods of measuring weight modulation. In preferred embodiments,the post-prandial response is measured in non-human animals, preferablymice. In preferred embodiments changes in dietary lipids are measured,preferably free fatty acids and/or triglycerides. In other embodiments,other physiologic parameters are measured including, but not limited to,levels of glucose, insulin, and leptin. In other preferred embodiments,free fatty acid oxidation is measured in cells in vitro or ex vivo,preferably in muscle cells or tissue of non-human animals, preferablymice. In yet other preferred embodiments weight modulation is measuredin human or non-human animals, preferably rodents (rats or mice),primates, canines, felines or procines on a high fat/sucrose diet.Optionally, “metabolic activity” includes other activities notspecifically identified herein. In general, “measurable parameters”relating to obesity and the field of metabolic research can be selectedfrom the group consisting of free fatty acid levels, free fatty acidoxidation, triglyceride levels, glucose levels, insulin levels, leptinlevels, food intake, weight, leptin and lipoprotein binding, uptake anddegradation and lipolysis stimulated receptor (LSR) expression.

In these metabolic assays, preferred KRIB polypeptides would cause asignificant change in at least one of the measurable parameters selectedfrom the group consisting of post-prandial lipemia, free fatty acidlevels, triglyceride levels, glucose levels, free fatty acid oxidation,and weight Alternatively, preferred KRIB polypeptides would have asignificant change in at least one of the measurable parameters selectedfrom the group consisting of an increase in LSR activity, an increase inleptin activity and an increase in lipoprotein activity. By “LSR”activity is meant expression of LSR on the surface of the cell, or in aparticular conformation, as well as its ability to bind, uptake, anddegrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor.

The invention is drawn, inter alia, to isolated, purified or recombinantKRIB polypeptides. KRIB polypeptides of the invention are useful forreducing or, using antagonists of KRIB polypeptides, increasing bodyweight either as a cosmetic treatment or for treatment or prevention ofmetabolic disorders. KRIB polypeptides are also useful inter alia inscreening assays for agonists or antagonists of KRIB polypeptideactivity; for raising KRIB polypeptide-specific antibodies; and indiagnostic assays. When used for cosmetic treatments, or for thetreatment or prevention of metabolic disorders, disorders or conditions,one or more KRIB polypeptide fragments can be provided to a subject.Thus, various fragments of the full-length protein can be combined intoa “cocktail” for use in the various treatment regimens.

All full-length KRIB polypeptides display a signal peptide.

The full-length KRIB-1 polypeptide displays a signal peptide about fromamino acids 1 to 20 of SEQ ID NO:2. The KRIB-1 mRNA is comprised of fourexons, which encode amino acids 1 to 51, amino acids 52 to 66, aminoacids 67 to 109 and amino acids 110 to 136 of SEQ ID NO: 2 respectively.

The full-length KRIB-2 polypeptide is comprised of about four distinctregions including:

-   -   1. an N-terminal putative signal peptide sequence about from        amino acids 1-14 of SEQ ID NO: 4;    -   2. a N-terminal Unique region about from amino acids 15-71 of        SEQ ID NO: 4;    -   3. a LAWN Domain region about from amino acids 72-94 of SEQ ID        NO: 4; and    -   4. a globular C-terminal unique region about from amino acids        95-97 of SEQ ID NO: 4.

The KRIB-2 mRNA is comprised of four exons, which encode amino acids 1to 21, amino acids 23 to 54, amino acids 55 to 94 and amino acids 96 and97 of SEQ ID NO: 4 respectively. The consensus sequence of the LAWNdomain is: GLLENLAWNLPNGPFSPNPDLLG.

The full-length KRIB-2R polypeptide is comprised of about four distinctregions including:

-   -   1. an N-terminal putative signal peptide sequence about from        amino acids 1-14 of SEQ ID NO: 6;    -   2. a N-terminal Unique region about from amino acids 15-38 of        SEQ ID NO: 6;    -   3. a LAWN Domain region about from amino acids 39-61 of SEQ ID        NO: 6; and    -   4. a globular C-terminal unique region about from amino acids        62-111 of SEQ ID NO: 6.

The full-length KRIB-3 polypeptide comprises an N-terminal putativesignal peptide sequence about from amino acids 1-18 of SEQ ID NO: 8.

The full-length KRIB-4 polypeptide is comprised of about three distinctregions including:

-   -   1. an N-terminal putative signal peptide sequence about from        amino acids 1-35 of SEQ ID NO: 10;    -   2. a central region about from amino acids 36-298 of SEQ ID NO:        10;    -   3. a leucine rich repeat C-terminal domain about from amino        acids 299-347 of SEQ ID NO: 10.

Furthermore, the central region about from amino acids 36-298 of SEQ IDNO: 10 includes:

-   -   1. eight leucine-rich repeats about from amino acids 93-116,        117-140, 141-164, 165-188, 189-212, 213-236,237-260 and 261-284        of SEQ ID NO:10.    -   2. eight segments exhibiting a periodic pattern in the        occurrence of leucine, proline, and asparagines about from amino        acids 84-107, 109-131, 132-155, 156-179, 180-203, 204-227,        228-251 and 252-275 of SEQ ID NO:10.

KRIB polypeptides of the invention include variants, fragments, analogsand derivatives of the KRIB polypeptides described above, includingmodified KRIB polypeptides. Preferred KRIB polypeptides are comprised ofat least one of the domains or segments described above. Preferred KRIBpolypeptides are comprised of at least one exon of the full-length orthe mature KRIB polypeptide.

The KRIB polypeptides of the present invention are preferably providedin an isolated form, and may be partially or substantially purified. Arecombinantly produced version of any one of the KRIB polypeptides canbe substantially purified by the one-step method described by Smith etal. ((1988) Gene 67:31-40) or by the methods described herein or knownin the art. Polypeptides of the invention also can be purified fromnatural or recombinant sources using antibodies directed against thepolypeptides of the invention by methods known in the art of proteinpurification.

Preparations of KRIB polypeptides of the invention involving a partialpurification of or selection for the KRIB polypeptides are alsospecifically contemplated. These crude preparations are envisioned to bethe result of the concentration of cells expressing KRIB polypeptideswith perhaps a few additional purification steps, but prior to completepurification of the fragment. The cells expressing KRIB polypeptides arepresent in a pellet, they are lysed, or the crude polypeptide islyophilized, for example.

KRIB polypeptide fragments can be any integer in length from at least 6consecutive amino acids to one amino acid less than a full-length KRIBpolypeptide. Thus, for the polypeptide of SEQ ID NO: 2, a KRIB-1polypeptide fragment can be any integer of consecutive amino acids from6 to 135, for example. For the polypeptide of SEQ ID NO: 4, a KRIB-2polypeptide fragment can be any integer of consecutive amino acids from6 to 96. For the polypeptide of SEQ ID NO: 8, a KRIB-3 polypeptidefragment can be any integer of consecutive amino acids from 6 to 211.For the polypeptide of SEQ ID NO: 10, a KRIB-4 polypeptide fragment canbe any integer of consecutive amino acids from 6 to 346, for example.The term “integer” is used herein in its mathematical sense and thusrepresentative integers include, but are not limited to: 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 238,339, 340, 341, 342, 343, 344, 345 and 346.

Each KRIB polypeptide fragment as described above can be furtherspecified in terms of its N-terminal and C-terminal positions. Forexample, every combination of a N-terminal and C-terminal position thatfragments of from 6 contiguous amino acids to one amino acid less thanthe full-length polypeptide of even SEQ ID Nos. 2-10 could occupy, onany given intact and contiguous full-length polypeptide sequence of evenSEQ ID Nos. 2-10 are included in the present invention. As a matter ofexample, a 6 consecutive amino acid fragment of KRIB-2 could occupypositions selected from the group consisting of 1-6, 2-7, 3-8, 4-9,5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20,16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30,26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40,36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50,46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60,56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70,66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80,76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90,86-91, 87-92, 88-93, 89-94, 90-95, 91-96, and 92-97 of a 97 consecutiveamino acid polypeptide. Similarly, the positions occupied by all theother fragments of sizes between 6 amino acids and 136, 97, 111, 212 and347 amino acids in even SEQ ID NOs: 2-10, respectively, are included inthe present invention and can also be immediately envisaged based onthese two examples and therefore, are not individually listed solely forthe purpose of not unnecessarily lengthening the specification.Furthermore, the positions occupied by fragments of 6 to 136, 97, 111,212 and 347 amino acids in even SEQ ID NOs: 2-10, respectively,consecutive amino acids in even SEQ ID NOs: 2-10 are included in thepresent invention and can also be immediately envisaged based on thesetwo examples and therefore are not individually listed solely for thepurpose of not unnecessarily lengthening the specification. In addition,the positions occupied by fragments of 6 consecutive amino acids to 1amino acid less than any other full-length KRIB polypeptide can also beenvisaged based on these two examples and therefore are not individuallylisted solely for the purpose of not unnecessarily lengthening thespecification.

The KRIB polypeptides of the present invention may alternatively bedescribed by the formula “n to c” (inclusive); where “n” equals theN-terminal most amino acid position (as defined by the sequence listing)and “c” equals the C-terminal most amino acid position (as defined bythe sequence listing) of the polypeptide; and further where “n” equalsan integer between 1 and the number of amino acids of the full-lengthpolypeptide sequence of the present invention minus 5; and where “c”equals an integer between 6 and the number of amino acids of thefull-length polypeptide sequence; and where “n” is an integer smallerthen “c” by at least 6. Therefore, for the sequences provided in SEQ IDNO: 2, “n” is any integer selected from the list consisting of: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 and “c” isany integer selected from the group consisting of: 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97. Everycombination of “n” and “c” positions are included as specificembodiments of the invention. Moreover, the formula “n” to “c” may bemodified as “‘n1-n2” to “c1-c2’”, wherein “n1-n2” and “c1-c2” representpositional ranges selected from any two integers above which representamino acid positions of the sequence listing. Alternative formulasinclude “‘n1-n2” to “c’” and “‘n” to “c1-c2’”. In a preferredembodiment, KRIB-1 polypeptide fragments of the invention may bedescribed by the formula where n=20 and c=136 of SEQ ID NO: 2, KRIB-2polypeptide fragments of the invention may be described by the formulawhere n=15 and c=97 of SEQ ID NO: 4, KRIB-2R polypeptide fragments ofthe invention may be described by the formula where n=15 and c=11 of SEQID NO: 6, KRIB-3 polypeptide fragments of the invention may be describedby the formula where n=18 and c=212 of SEQ ID NO: 8, and KRIB-4polypeptide fragments of the invention may be described by the formulawhere n=18 and c=287 of SEQ ID NO: 10.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast”, “equal to”, “equal to or less than”, “less than”, “at least______ but not greater than ______” or “from ______ to ______” aspecified size or specified N-terminal and/or C-terminal positions. Itis noted that all ranges used to describe any embodiment of the presentinvention are inclusive unless specifically set forth otherwise.

The present invention also provides for the exclusion of any individualfragment specified by N-terminal and C-terminal positions or of anyfragment specified by size in amino acid residues as described above.Further, any number of fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above may makeup a polypeptide fragment in any combination and may optionally includenon-KRIB polypeptide sequences as well.

In other preferred embodiments, said KRIB-1 polypeptide fragments havingactivity selected from the group consisting of prevention of weightgain, weight reduction, and maintenance of weight loss are selected fromamino acids 20-136, 21-136, 22-136, 23-136, 24-136, 25-136, 26-136,27-136, 28-136, 29-136, 30-136, 31-136, 32-136, 33-136, 34-136, 35-136,36-136, 37-136, 38-136, 39-136, 40-136, 41-136, 42-136, 43-136, 44-136,45-136, 46-136, 47-136, 48-136, 49-136, 50-136, 51-136, 52-136, 53-136,54-136, 55-136, 56-136, 57-136, 58-136, 59-136, 60-136, 61-136, 62-136,63-136, 64-136, 65-136, 66-136, 67-136, 68-136, 69-136, 70-136, 71-136,72-136, 73-136, 74-136, 75-136, 76-136, 77-136, 78-136, 79-136, 80-136,81-136, 82-136, 83-136, 84-136, 85-136, 86-136, 87-136, 88-136, 89-136,90-136, 91-136, 92-36, 93-136, 94-136, 95-136, 96-136, 97-136, 98-136,99-136, 100-136, 101-136, 102-136, 103-136, 104-136, 105-136, 106-136,107-136, 108-136, 109-136, 110-136, 111-136, 112-136, 113-136, 114-136,115-136, 116-136, 117-136, 118-136, 119-136, 120-136, 121-136, 122-136,123-136, 124-136, 125-136, 126-136, 127-136, 128-136, 129-136 or 130-136of SEQ ID NO: 2, where it is understood that amino acid 20 is taken torepresent the N-terminal amino acid of mature KRIB-1 polypeptide absentthe signal peptide.

In other preferred embodiments, said KRIB-2 and KRIB-2R polypeptidefragments having activity selected from the group consisting ofprevention of weight gain, weight reduction, and maintenance of weightloss are selected from amino acids 15-97, 16-97, 17-97, 18-97, 19-97,20-97, 21-97, 22-97, 23-97, 24-97, 25-97, 26-97, 27-97, 28-97, 29-97,30-97, 31-97, 32-97, 33-97, 34-97, 35-97, 36-97, 37-97, 38-97, 39-97,40-97, 41-97, 42-97, 43-97, 44-97, 45-97, 46-97, 47-97, 48-97, 49-97,50-97, 51-97, 52-97, 53-97, 54-97, 55-97, 56-97, 57-97, 58-97, 59-97,60-97, 61-97, 62-97, 63-97, 64-97, 65-97, 66-97, 67-97, 68-97, 69-97,70-97, 71-97, 72-97, 73-97, 74-97, 75-97, 76-97, 77-97, 78-97, 79-97,80-97, 81-97, 82-97, 83-97, 84-97, 85-97, 86-97, 87-97, 88-97, 89-97,90-97, or 91-97 of SEQ ID NO: 4 where it is understood that amino acid15 is taken to represent the N-terminal amino acid of mature KRIB-2 andKRIB-2R polypeptide absent the signal peptide.

In other preferred embodiments, KRIB-2R polypeptide fragments havingactivity are selected from amino acids 15-111, 16-111, 17-111, 18-111,19-111, 20-111, 21-111, 22-111, 23-111, 24-111, 25-111, 26-111, 27-111,28-111, 29-111, 30-111, 31-111, 32-111, 33-111, 34-111, 35-111, 36-111,37-111, 38-111, 39-111, 40-111, 41-111, 42-111, 43-111, 44-111, 45-111,46-111, 47-111, 48-111, 49-111, 50-111, 51-111, 52-111, 53-111, 54-111,55-111, 56-111, 57-111, 58-111, 59-111, 60-111, 61-111, 62-111, 63-111,64-111, 65-111, 66-111, 67-111, 68-111, 69-111, 70-111, 71-111, 72-111,73-111, 74-111, 75-111, 76-111, 77-111, 78-111, 79-111, 80-111, 81-111,82-111, 83-111, 84-111, 85-111, 86-111, 87-111, 88-111, 89-111, 90-111,91-111, 92-111, 93-111, 94-111, 95-111, 96-111, 97-111, 98-111, 99-111,100-111, 101-111, 102-111, 103-111, 104-111, or 105-111 of SEQ ID NO: 6where it is understood that amino acid 15 is taken to represent theN-terminal amino acid of mature KRIB-2R polypeptide absent the signalpeptide.

In other preferred embodiments, said KRIB-2 and KRIB-2R polypeptidefragments having activity selected from the group consisting ofprevention of weight gain, weight reduction, and maintenance of weightloss comprise the LAWN Domain region about from amino acids 72-94 of SEQID NO: 4 or from about amino acids 39-61 of SEQ ID NO: 6.

In preferred embodiments, KRIB-3 polypeptide fragments having activityselected from the group consisting of lipid partitioning, lipidmetabolism, and insulin-like activity are selected from amino acids18-212, 19-212, 20-212, 21-212, 22-212, 23-212, 24-212, 25-212, 26-212,27-212, 28-212, 29-212, 30-212, 31-212, 32-212, 33-212, 34-212, 35-212,36-212, 37-212, 38-212, 39-212, 40-212, 41-212, 42-212, 43-212, 44-212,45-212, 46-212, 47-212, 48-212, 49-212, 50-212, 51-212, 52-212, 53-212,54-212, 55-212, 56-212, 57-212, 58-212, 59-212, 60-212, 61-212, 62-212,63-212, 64-212, 65-212, 66-212, 67-212, 68-212, 69-212, 70-212, 71-212,72-212, 73-212, 74-212, 75-212, 76-212, 77-212, 78-212, 79-212, 80-212,81-212, 82-212, 83-212, 84-212, 85-212, 86-212, 87-212, 88-212, 89-212,90-212, 91-212, 92-212, 93-212, 94-212, 95-212, 96-212, 97-212, 98-212,99-212, 100-212, 101-212, 102-212, 103-212, 104-212, 105-212, 106-212,107-212, 108-212, 109-212, 110-212, 111-212, 112-212, 113-212, 114-212,115-212, 116-212, 117-212, 118-212, 119-212, 120-212, 121-212, 122-212,123-212, 124-212, 125-212, 126-212, 127-212, 128-212, 129-212, 130-212,131-212, 132-212, 133-212, 134-212, 135-212, 136-212, 137-212, 138-212,139-212, 140-212, 141-212, 142-212, 143-212, 144-212, 145-212, 146-212,147-212, 148-212, 149-212, 150-212, 151-212, 152-212, 153-212, 154-212,155-212, 156-212, 157-212, 158-212, 159-212, 160-212, 161-212, 162-212,163-212, 164-212, 165-212, 166-212, 167-212, 168-212, 169-212, 170-212,171-212, 172-212, 173-212, 174-212, 175-212, 176-212, 177-212, 178-212,179-212, 180-212, 181-212, 182-212, 183-212, 184-212, 185-212, 186-212,187-212, 188-212, 189-212, 190-212, 191-212, 192-212, 193-212, 194-212,195-212, 196-212, 197-212, 198-212, 199-212, 200-212, 201-212, 202-212,203-212, 204-212, 205-212, 206-212 or 207-212 of SEQ ID NO: 8, where itis understood that amino acid 18 is taken to represent the N-terminalamino acid of mature KRIB-3 polypeptide absent the signal peptide.

In preferred embodiments, KRIB-4 polypeptide fragments comprising all orpart of (i) one of the LRR domains; (ii) one of the segments exhibitinga periodic pattern in the occurrence of leucine, proline, andasparagines; or (iii) the LRRCT domain and having activity selected fromthe group consisting of lipid partitioning, lipid metabolism, andinsulin-like activity are selected from amino acids 36-347, 36-107,36-131, 36-155, 36-179, 36-203, 36-227, 36-251, 36-275, 36-116, 36-140,36-164, 36-188, 36-212, 36-236, 36-260, 36-284, 84-107, 109-131,132-155, 156-179, 180-203, 204-227, 228-251, 252-275, 84-131, 84-155,84-179, 84-203, 84-227, 84-251, 84-275, 109-155, 109-179, 109-203,109-227, 109-251, 109-275, 132-179, 132-203, 132-227, 132-251,132-275,156-203, 156-227, 156-251, 156-275, 180-227, 180-251, 180-275,204-251,204-275,228-275, 93-116, 117-140, 141-164, 165-188, 189-212, 213-236,237-260, 261-284, 93-140, 93-164, 93-188, 93-212, 93-236, 93-260,93-284, 117-164, 117-188, 117-212, 117-236, 117-260, 117-284, 141-188,141-212, 141-236, 141-260, 141-284, 165-212, 165-236, 165-260,165-284,189-236,189-260,189-284,213-260,213-284,237-284, 299-347,93-347,117-347,141-347, 165-347, 189-347,213-347, 237-347, 261-347,84-347, 109-347, 132-347, 156-347, 180-347, 204-347, 228-347 and 252-347of SEQ ID NO: 10, where it is understood that amino acid 36 is taken torepresent the N-terminal amino acid of mature KRIB-4 polypeptide absentthe signal peptide.

Other preferred are KRIB-4 polypeptide fragments having activity andcomprising all or part of one of the LRR domains that are selected fromamino acids 36-347, 36-116, 36-140, 36-164, 36-188, 36-212, 36-236,36-260, 36-284, 93-116, 117-140, 141-164, 165-188, 189-212, 213-236,237-260, 261-284, 93-140, 93-164, 93-188, 93-212, 93-236, 93-260,93-284, 117-164, 117-188, 117-212, 117-236, 117-260, 117-284, 141-188,141-212, 141-236, 141-260, 141-284, 165-212, 165-236, 165-260, 165-284,189-236, 189-260, 189-284, 213-260, 213-284 and 237-284 of SEQ ID NO:10, where it is understood that amino acid 36 is taken to represent theN-terminal amino acid of mature KRIB-4 polypeptide absent the signalpeptide.

Other particularly preferred are KRIB-4 polypeptide fragments havingactivity and comprising all or part of one of the segments exhibiting aperiodic pattern in the occurrence of leucine, proline, and asparaginesthat are selected from amino acids 36-347, 36-107, 36-131, 36-155,36-179, 36-203, 36-227, 36-251, 36-275, 84-107, 109-131, 132-155,156-179, 180-203, 204-227, 228-251, 252-275, 84-131, 84-155, 84-179,84-203, 84-227, 84-251, 84-275, 109-155, 109-179, 109-203, 109-227,109-251, 109-275, 132-179, 132-203, 132-227, 132-251, 132-275, 156-203,156-227, 156-251, 156-275, 180-227, 180-251, 180-275, 204-251, 204-275and 228-275 of SEQ ID NO: 10, where it is understood that amino acid 36is taken to represent the N-terminal amino acid of mature KRIB-4polypeptide absent the signal peptide.

Other preferred are KRIB-4 polypeptide fragments having activity andcomprising all or part of the LRRCT domain that are selected from aminoacids 36-347, 299-347, 93-347, 117-347, 141-347, 165-347, 189-347,213-347, 237-347, 261-347, 84-347, 109-347, 132-347, 156-347, 180-347,204-347, 228-347 and 252-347 of SEQ ID NO: 10, where it is understoodthat amino acid 36 is taken to represent the N-terminal amino acid ofmature KRIB-4 polypeptide absent the signal peptide.

Regulated proteolytic cleavage of full-length KRIB polypeptides of theinvention in vivo is believed by the inventors to lead to the effectivegeneration in vivo of KRIB polypeptide fragments of the invention havingactivity selected from the group consisting of lipid partitioning, lipidmetabolism, or insulin-like activity. Without wishing to be bound by anyparticular theory, particularly preferred KRIB polypeptide fragments ofthe invention having activity selected from the group consisting oflipid partitioning, lipid metabolism, and insulin-like activity arefragments of KRIB polypeptide of even SEQ ID NOs: 2-10 generated byproteolytic cleavage. Without wishing to be bound by any particulartheory, other particularly preferred KRIB polypeptide fragments of theinvention having activity selected from the group consisting ofprevention of weight gain, weight reduction, and maintenance of weightloss are fragments of KRIB polypeptide of even SEQ ID NOs: 2-10generated by proteolytic cleavage.

Without wishing to be bound by any particular theory, also particularlypreferred are KRIB polypeptide fragments having activity as describedabove and capable of interacting with TGF-beta I type II receptors.

Without wishing to be bound by any particular theory, also particularlypreferred are KRIB polypeptide fragments having activity as describedabove and not capable of interacting with TGF-beta I type II receptors.

KRIB polypeptides of the invention include variants, fragments, analogsand derivatives of the KRIB polypeptides described above, includingmodified KRIB polypeptides.

Variants

It will be recognized by one of ordinary skill in the art that someamino acids of the KRIB polypeptide sequences of the present inventioncan be varied without significant effect on the structure or function ofthe proteins; there will be critical amino acids in the sequence thatdetermine activity. Thus, the invention further includes variants ofKRIB polypeptides that have metabolic activity as described above. Suchvariants include KRIB polypeptide sequences with one or more amino aciddeletions, insertions, inversions, repeats, and substitutions eitherfrom natural mutations or human manipulation selected according togeneral rules known in the art so as to have little effect on activity.Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided below.

There are two main approaches for studying the tolerance of an aminoacid sequence to change (see, Bowie, et al. (1990) Science, 247,1306-10). The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions and indicate which amino acid changes arelikely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein.

In the case of an amino acid substitution in the amino acid sequence ofa polypeptide according to the invention, one or several amino acids canbe replaced by “equivalent” amino acids. The expression. “equivalent”amino acid is used herein to designate any amino acid that may besubstituted for one of the amino acids having similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 1 Original Residue ExemplarySubstitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R)lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C)ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H)asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leuLeu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr, ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of theKRIB polypeptides are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., NuclAcids Res, 13:4331 (1986); Zoller et al., Nucl Acids Res, 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the KRIB variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Amino acids in the KRIB polypeptide sequences of the invention that areessential for function can also be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham, et al. (1989) Science 244:1081-5). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for metabolicactivity using assays as described above. Of special interest aresubstitutions of charged amino acids with other charged or neutral aminoacids that may produce proteins with highly desirable improvedcharacteristics, such as less aggregation. Aggregation may not onlyreduce activity but also be problematic when preparing pharmaceutical orphysiologically acceptable formulations, because aggregates can beimmunogenic (see, e.g., Pinckard, et al., (1967) Clin Exp Immunol2:331-340; Robbins, et al., (1987) Diabetes 36:838-41; and Cleland, etal., (1993) Crit Rev Ther Drug Carrier Syst 10:307-77).

Thus, the fragment, derivative, analog, or homolog of the KRIBpolypeptides of the present invention may be, for example: (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code (i.e. may be a non-naturallyoccurring amino acid); or (ii) one in which one or more of the aminoacid residues includes a substituent group; or (iii) one in which theKRIB polypeptides are fused with another compound, such as a compound toincrease the half-life of the fragment (for example, polyethyleneglycol); or (iv) one in which the additional amino acids are fused tothe above form of the fragment, such as an IgG Fc fusion region peptideor leader or secretory sequence or a sequence which is employed forpurification of the above form of the fragment or a pro-proteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of KRIB polypeptides having an aminoacid sequence which contains at least one conservative amino acidsubstitution, but not more than 50 conservative amino acidsubstitutions, not more than 40 conservative amino acid substitutions,not more than 30 conservative amino acid substitutions, and not morethan 20 conservative amino acid substitutions. Also provided arepolypeptides which comprise the amino acid sequence of a KRIB fragment,having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

In addition, amino acids have chirality within the body of either L orD. In some embodiments it is preferable to alter the chirality of theamino acids in the KRIB polypeptide fragments of the invention in orderto extend half-life within the body. Thus, in some embodiments, one ormore of the amino acids are preferably in the L configuration. In otherembodiments, one or more of the amino acids are preferably in the Dconfiguration.

Percent Identity

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 50% identical, at least 60%identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to a KRIB polypeptide as described above. By apolypeptide having an amino acid sequence at least, for example, 95%“identical” to a KRIB polypeptide amino acid sequence is meant that theamino acid sequence is identical to the KRIB polypeptide sequence exceptthat it may include up to five amino acid alterations per each 100 aminoacids of the KRIB polypeptide amino acid sequence. The referencesequence is the KRIB polypeptide with a sequence corresponding to thesequence provided in even SEQ ID NOs: 2-10. Thus, to obtain apolypeptide having an amino acid sequence at least 95% identical to apolypeptide amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the sequence may be inserted, deleted, or substituted withanother amino acid compared with the KRIB polypeptide sequence. Thesealterations may occur at the amino or carboxy termini or anywherebetween those terminal positions, interspersed either individually amongresidues in the sequence or in one or more contiguous groups within thesequence.

As a practical matter, whether any particular polypeptide is apercentage identical to a KRIB polypeptide can be determinedconventionally using known computer programs. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl AcadSci USA 85:2444-8; Altschul et al., (1990) J Mol Biol 215:403-410;Thompson et al., (1994) Nucleic Acids Res 22(2):4673-4680; Higgins etal, (1996) Meth Enzymol 266:383-402; Altschul et al, (1997) NucleicAcids Res 25:3389-3402; Altschul et al., (1993) Nature Genetics3:266-272). In a particularly preferred embodiment, protein and nucleicacid sequence homologies are evaluated using the Basic Local AlignmentSearch Tool (“BLAST”), which is well known in the art (See, e.g., Karlinand Altschul (1990) Proc Natl Acad Sci USA 87.2264-8; Altschul et al.,1990, 1993, 1997, all supra). In particular, five specific BLASTprograms are used to perform the following tasks:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnetet al., (1992) Science 256:1443-5; Henikoff and Henikoff (1993) Proteins17:49-61). Less preferably, the PAM or PAM250 matrices may also be used(See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for DetectingDistance Relationships: Atlas of Protein Sequence and Structure,Washington: National Biomedical Research Foundation). The BLAST programsevaluate the statistical significance of all high-scoring segment pairsidentified, and preferably selects those segments which satisfy auser-specified threshold of significance, such as a user-specifiedpercent homology. Preferably, the statistical significance of ahigh-scoring segment pair is evaluated using the statisticalsignificance formula of Karlin (See, e.g., Karlin and Altschul, (1990)Proc Natl Acad Sci USA 87:2264-8). The BLAST programs may be used withthe default parameters or with modified parameters provided by the user.Preferably, the parameters are default parameters.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (1990) Comp App Biosci 6:237-245. In a sequence alignmentthe query and subject sequences are both amino acid sequences. Theresult of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB amino acid alignment are:Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected because the FASTDB programdoes not account for N- and C-terminal truncations of the subjectsequence when calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,that are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what is used for the purposes of the present invention. Onlyresidues to the N- and C-termini of the subject sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryamino acid residues outside the farthest N- and C-terminal residues ofthe subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100-residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.

In another example, a 90-residue subject sequence is compared with a100-residue query sequence. This time the deletions are internal sothere are no residues at the N- or C-termini of the subject sequence,which are not matched/aligned with the query. In this case, the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected. No othermanual corrections are made for the purposes of the present invention.

Production

Note, throughout the disclosure, wherever KRIB polypeptides arediscussed, KRIB fragments, variants and derivatives are specificallyintended to be included as a preferred subset of KRIB polypeptides.

KRIB polypeptides are preferably isolated from human or mammalian tissuesamples or expressed from human or mammalian genes in human or mammaliancells. The KRIB polypeptides of the invention can be made using routineexpression methods known in the art. The polynucleotide encoding thedesired polypeptide is ligated into an expression vector suitable forany convenient host. Both eukaryotic and prokaryotic host systems areused in forming recombinant polypeptides. The polypeptide is thenisolated from lysed cells or from the culture medium and purified to theextent needed for its intended use. Purification is by any techniqueknown in the art, for example, differential extraction, saltfractionation, chromatography, centrifugation, and the like. See, forexample, Methods in Enzymology for a variety of methods for purifyingproteins.

In an alternative embodiment, the polypeptides of the invention areisolated from milk. The polypeptides can be purified as full-length KRIBpolypeptides, which can then be cleaved, if appropriate, in vitro togenerate a KRIB fragment, or, alternatively, KRIB fragments themselvescan be purified from the milk. Any of a large number of methods can beused to purify the present polypeptides from milk, including thosetaught in Protein Purification Applications, A Practical Approach (NewEdition), Edited by Simon Roe, AEA Technology Products and Systems,Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia3:337-50; Wilkins and Velander (1992) 49:333-8; U.S. Pat. Nos.6,140,552; 6,025,540; Hennighausen, Protein Expression and Purification,vol. 1, pp. 3-8 (1990); Harris et al. (1997) Bioseparation 7:31-7;Degener et al. (1998) J Chromatog 799:125-37; Wilkins (1993) J CellBiochem. Suppl. 0 (17 part A):39; the entire disclosures of each ofwhich are herein incorporated by reference. In a typical embodiment,milk is centrifuged, e.g. at a relatively low speed, to separate thelipid fraction, and the aqueous supernatant is then centrifuged at ahigher speed to separate the casein in the milk from the remaining,“whey” fraction. Often, biomedical proteins are found in this wheyfraction, and can be isolated from this fraction using standardchromatographic or other procedures commonly used for proteinpurification, e.g. as described elsewhere in the present application. Inone preferred embodiment, KRIB polypeptides are purified usingantibodies specific to KRIB polypeptides, e.g. using affinitychromatography. In addition, methods can be used to isolate particularKRIB fragments, e.g. electrophoretic or other methods for isolatingproteins of a particular size. The KRIB polypeptides isolating usingthese methods can be naturally occurring, as KRIB polypeptides have beendiscovered to be naturally present in the milk of mammals, or can be theresult of the recombinant production of the protein in the mammaryglands of a non-human mammal, as described infra. In one suchembodiment, the KRIB is produced as a fusion protein with aheterologous, antigenic polypeptide sequence, which antigenic sequencecan be used to purify the protein, e.g., using standard immuno-affinitymethodology.

In addition, shorter protein fragments may be produced by chemicalsynthesis. Alternatively, the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

Any KRIB cDNA including that in odd SEQ ID NOs 1-9 can be used toexpress KRIB polypeptides. The nucleic acid encoding the KRIBpolypeptide to be expressed is operably linked to a promoter in anexpression vector using conventional cloning technology. The KRIB cDNAinsert in the expression vector may comprise the coding sequence for:the full-length KRIB polypeptide; from 6 amino acids to one amino acidless than the full-length KRIB polypeptide; a KRIB polypeptide fragment;or variants and % similar polypeptides.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art, some of which aredescribed herein. Commercially available vectors and expression systemsare available from a variety of suppliers including Genetics Institute(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison,Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhanceexpression and facilitate proper protein folding, the codon context andcodon pairing of the sequence can be optimized for the particularexpression organism into which the expression vector is introduced, asexplained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosuresof which are incorporated by reference herein in their entirety.

If the nucleic acid encoding any one of the KRIB polypeptides lacks amethionine to serve as the initiation site, an initiating methionine canbe introduced next to the first codon of the nucleic acid usingconventional techniques. Similarly, if the insert from the KRIBpolypeptide cDNA lacks a poly A signal, this sequence can be added tothe construct by, for example, splicing out the Poly A signal from pSG5(Stratagene) using BglI and SalI restriction endonuclease enzymes andincorporating it into the mammalian expression vector pXT1 (Stratagene).pXT1 contains the LTRs and a portion of the gag gene from Moloney MurineLeukemia Virus. The position of the LTRs in the construct allowsefficient stable transfection. The vector includes the Herpes SimplexThymidine Kinase promoter and the selectable neomycin gene.

The nucleic acid encoding KRIB can be obtained by PCR from a vectorcontaining the KRIB nucleotide sequence using oligonucleotide primerscomplementary to the desired KRIB polynucleotide and containingrestriction endonuclease sequences for Pst I incorporated into the5′primer and BglII at the 5′ end of the corresponding cDNA 3′primer,taking care to ensure that the sequence encoding the KRIB is positionedproperly with respect to the poly A signal. The purified polynucleotideobtained from the resulting PCR reaction is digested with PstI, bluntended with an exonuclease, digested with BglII, purified and ligated topXT1, now containing a poly A signal and digested with BglII.

Transfection of a KRIB expressing vector into mouse NIH 3T3 cells is oneembodiment of introducing polynucleotides into host cells. Introductionof a polynucleotide encoding a polypeptide into a host cell can beeffected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, or other methods. Such methods are described inmany standard laboratory manuals, such as Davis et al. ((1986) Methodsin Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).It is specifically contemplated that the polypeptides of the presentinvention may in fact be expressed by a host cell lacking a recombinantvector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. Preferably said glycosylation may occur at one or morehydroxylated lysine residues within the collagen-like region. Preferablythe polypeptides of the invention are non-glycosylated. In addition,polypeptides of the invention may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination, see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,(1989) Proc Natl Acad Sci USA 86:8932-5; Koller et al., (1989) Proc NatlAcad Sci USA 86:8927-31; and Zijlstra et al. (1989) Nature 342:435-8;the disclosures of each of which are incorporated by reference in theirentireties).

Modifications

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (See, e.g., Creighton, 1983 Proteins.New York, N.Y.: W.H. Freeman and Company; and Hunkapiller et al., (1984)Nature 310:105-11). For example, a relative short fragment of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thefragment sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the polypeptide.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention that may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity. See U.S. Pat. No. 4,179,337.The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the polypeptide with consideration of effects on functionalor antigenic domains of the polypeptide. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al. (1992) Exp Hematol (8):1028-35, reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to, protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus may be accomplished by reductive alkylation,which exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

Multimers

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,pharmaceutical or physiologically acceptable compositions) containingthem. In specific embodiments, the polypeptides of the invention aremonomers, dimers, trimers or tetramers. In additional embodiments, themultimers of the invention are at least dimers, at least trimers, or atleast tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the KRIB polypeptides of the invention(including polypeptide fragments, variants, splice variants, and fusionproteins corresponding to these polypeptide fragments as describedherein). These homomers may contain polypeptide fragments havingidentical or different amino acid sequences. In a specific embodiment, ahomomer of the invention is a multimer containing only polypeptidefragments having an identical amino acid sequence. In another specificembodiment, a homomer of the invention is a multimer containingpolypeptide fragments having different amino acid sequences. In specificembodiments, the multimer of the invention is a homotrimer (e.g.,containing polypeptides having identical and/or different amino acidsequences). In other specific embodiments, the multimer of the inventionis a homohexamer (e.g., containing polypeptides having identical and/ordifferent amino acid sequences). In preferred specific embodiments, themultimer of the invention is a homotrimer containing only polypeptidefragments having an identical amino acid sequence. In other preferredspecific embodiments, the multimer of the invention is a homohexamercontaining only polypeptide fragments having an identical amino acidsequence. In additional embodiments, the homomeric multimer of theinvention is at least a homodimer, at least a homotrimer, at least ahomotetramer, or at least a homohexamer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., corresponding to differentproteins or polypeptides thereof) in addition to the polypeptides of theinvention. In a specific embodiment, the multimer of the invention is aheterotrimer. In other specific embodiment, the multimer of theinvention is a heterohexamer. In additional embodiments, the heteromericmultimer of the invention is at least a heterodimer, at least aheterotrimer, at least a heterotetramer, or at least a heterohexamer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences,which interact in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins, and have since been found ina variety of different proteins (Landschulz et al., (1988) Genes Dev2:786-800). Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. FEBS Letters (1994) 344:191-5 and inU.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention. In another example, proteins of the invention are associatedby interactions between Flag® & polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, proteins of the invention are associated byinteractions between heterologous polypeptide sequence contained inFlag® fusion proteins of the invention and anti Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, at least 30techniques known in the art may be applied to generate liposomescontaining the polypeptide components desired to be contained in themultimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (See, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

II. KRIB Polynucleotides of the Invention

Preferred polynucleotides are those that encode KRIB polypeptides of theinvention. The recombinant polynucleotides encoding KRIB polypeptidescan be used in a variety of ways, including, but not limited to,expressing the polypeptides in recombinant cells for use in screeningassays for antagonists and agonists of its activity as well as tofacilitate its purification for use in a variety of ways including, butnot limited to screening assays for agonists and antagonists of itsactivity, diagnostic screens, and raising antibodies, as well astreatment and/or prevention of metabolic disorders and/or to reduce bodymass.

The invention relates to the polynucleotides encoding KRIB polypeptidesand variant polypeptides thereof as described herein. Thesepolynucleotides may be purified, isolated, and/or recombinant. In allcases, the desired KRIB polynucleotides of the invention are those thatencode. KRIB polypeptides of the invention having metabolic activity asdescribed and discussed herein.

Fragments

A polynucleotide fragment is a polynucleotide having a sequence thatentirely is the same as part, but not all, of the full-length KRIBpolypeptide or a specified KRIB polypeptide nucleotide sequence. Suchfragments may be “free-standing”, i.e. not part of or fused to otherpolynucleotides, or they may be comprised within another non-KRIB(heterologous) polynucleotide of which they form a part or region.However, several KRIB polynucleotide fragments may be comprised within asingle polynucleotide.

The KRIB polynucleotides of the invention comprise from 18 consecutivebases to the full-length polynucleotide sequences encoding the intactKRIB polypeptide, for example the full-length KRIB polypeptidepolynucleotide sequences in odd SEQ ID NOs: 1-9. In one aspect of thisembodiment, the polynucleotide comprises at least in one aspect of thisembodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400,405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540,545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610,615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750,755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890,895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960,965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025,1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085,1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145,1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205,1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265,1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325,1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385,1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445,1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495, 1500, 1505,1510, 1515, 1520, 1525, 1530, 1535, 1540, 1545, 1550, 1555, 1560, 1565,1570, 1575, 1580, 1585, 1590, 1595, 1600, 1605, 1610, 1615, 1620, 1625,1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685,1690, 1695, 1700, 1705, 1710, 1715, 1720, 1725, 1730, 1735, 1740, 1745,1750, 1755, 1760, 1765, 1770, 1775, 1780, 1785, 1790, 1795, 1800 or 1801consecutive nucleotides of a polynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer representing the 3′most nucleotide position of the intact KRIB polypeptide cDNA as setforth in odd SEQ ID NOs: 1-9 or elsewhere herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG)with the remaining nucleotides numbered consecutively. Therefore, everycombination of a 5′ and 3′ nucleotide position that a polynucleotidefragment, at least 18 contiguous nucleotides in length, could occupy onan intact KRIB polypeptide encoding a polynucleotide of the presentinvention is included in the invention as an individual species. Thepolynucleotide fragments specified by 5′ and 3′ positions can beimmediately envisaged and are therefore not individually listed solelyfor the purpose of not unnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 19 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18 nucleotides;and where “x” is an integer less than “y” by at least 18.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above.

The KRIB polynucleotide fragments of the invention comprise from 18consecutive bases to the full-length polynucleotide sequence encodingthe KRIB polypeptide fragments described in Section II of the PreferredEmbodiments of the Invention. In one aspect of this embodiment, thepolynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485,490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, or 783 consecutive nucleotides of a polynucleotide of thepresent invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer corresponding to the3′ most nucleotide position of a KRIB fragment cDNA herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the open reading frame (ORF), i.e., the nucleotide “A” ofthe start codon (ATG) with the remaining nucleotides numberedconsecutively. Therefore, every combination of a 5′ and 3′ nucleotideposition that a polynucleotide fragment invention, at least 18contiguous nucleotides in length, could occupy on a KRIB fragmentpolynucleotide of the present invention is included in the invention asan individual species. The polynucleotide fragments specified by 5′ and3′ positions can be immediately envisaged and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of the KRIBpolynucleotide sequences of the present invention minus 18, and where“y” equals an integer between 9 and the number of nucleotides of theKRIB polynucleotide sequences of the present invention; and where “x” isan integer smaller than “y” by at least 18. Every combination of “x” and“y” positions are included as specific embodiments of the invention.Moreover, the formula “x” to “y” may be modified as “‘x1-x2” to“y1-y2’”, wherein “x1-x2” and “y1-y2” represent positional rangesselected from any two nucleotide positions of the sequence listing.Alternative formulas include “‘x1-x2” to “y’” and “‘x” to “y1-y2’”.

These specific embodiments, and other polynucleotide fragmentembodiments described herein may be modified as being “at least”, “equalto”, “equal to or less than”, “less than”, “at least ______ but notgreater than ______ ” or “from ______ to ______”. a specified size orspecified 5′ and/or 3′ positions.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above.

Variants

In other preferred embodiments, variants of KRIB polynucleotidesencoding KRIB polypeptides are envisioned. Variants of polynucleotides,as the term is used herein, are polynucleotides whose sequence differsfrom a reference polynucleotide. A variant of a polynucleotide may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

Polynucleotide variants that comprise a sequence substantially differentfrom those described above but that, due to the degeneracy of thegenetic code, still encode KRIB polypeptides of the present inventionare also specifically envisioned. It would also be routine for oneskilled in the art to generate the degenerate variants described above,for instance, to optimize codon expression for a particular host (e.g.,change codons in the human mRNA to those preferred by other mammalian orbacterial host cells).

As stated above, variant polynucleotides may occur naturally, such as anatural allelic variant, or by recombinant methods. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism (See, e.g., B. Lewin,(1990) Genes IV, Oxford University Press, New York). Non-naturallyoccurring variants may be produced using art-known mutagenesistechniques. Such nucleic acid variants include those produced bynucleotide substitutions, deletions, or additions. The substitutions,deletions, or additions may involve one or more nucleotides. Alterationsin the coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of KRIB polypeptides of theinvention. Also preferred in this regard are conservative substitutions.

Nucleotide changes present in a variant polynucleotide are preferablysilent, which means that they do not alter the amino acids encoded bythe polynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence.

In cases where the nucleotide substitutions result in one or more aminoacid changes, preferred KRIB polypeptides include those that retain oneor more metabolic activity as described in Section I of the PreferredEmbodiments of the Invention.

By “retain the same activities” is meant that the activity measuredusing the polypeptide encoded by the variant KRIB polynucleotide inassays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%,and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125%of the activity measured using a KRIB polypeptide described in theExamples Section herein.

By the activity being “increased” is meant that the activity measuredusing the polypeptide encoded by the variant KRIB polynucleotide inassays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%,180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%,or 500% of the activity measured using a KRIB polypeptide described inthe Examples Section herein.

By the activity being “decreased” is meant that the activity measuredusing the polypeptide encoded by the variant KRIB polynucleotide inassays is decreased by at least 25%, 30%, 35%, 40%, 45%, 50%, 75%, 80%,90% or 95% of the activity measured using a KRIB polypeptide describedin the Examples Section herein

Percent Identity

The present invention is further directed to nucleic acid moleculeshaving sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or99% identical to the polynucleotide sequences of odd SEQ ID NOs: 1-9 orfragments thereof that encode a polypeptide having metabolic activity asdescribed in Section I of the Preferred Embodiments of the Invention. Ofcourse, due to the degeneracy of the genetic code, one of ordinary skillin the art will immediately recognize that a large number of the nucleicacid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or99% identical to the nucleic acid sequences shown in odd SEQ ID NOs: 1-9or fragments thereof will encode a polypeptide having biologicalactivity. In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode a polypeptide having biological activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly affectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described previously in Section I ofthe Preferred Embodiments of the Invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the KRIBpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted, inserted, or substituted with another nucleotide. The querysequence may be an entire sequence or any fragment specified asdescribed herein.

The methods of determining and defining whether any particular nucleicacid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to a nucleotide sequence of the presentinvention can be done by using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al.,((1990) Comput Appl Biosci. July; 6(3):237-45). In a sequence alignmentthe query and subject sequences are both DNA sequences. An RNA sequencecan be compared by first converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90-nucleotide subject sequence is aligned to a100-nucleotide query sequence to determine percent identity. Thedeletions occur at the 5′ end of the subject sequence and therefore, theFASTDB alignment does not show a matched/alignment of the first 10nucleotides at 5′ end. The 10 unpaired nucleotides represent 10% of thesequence (number of nucleotides at the 5′ and 3′ ends not matched/totalnumber of nucleotides in the query sequence) so 10% is subtracted fromthe percent identity score calculated by the FASTDB program. If theremaining 90 nucleotides were perfectly matched the final percentidentity would be 90%.

In another example, a 90 nucleotide subject sequence is compared with a100 nucleotide query sequence. This time the deletions are internaldeletions so that there are no nucleotides on the 5′ or 3′ of thesubject sequence which are not matched/aligned with the query. In thiscase the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made for thepurposes of the present invention.

Fusions

Further included in the present invention are polynucleotides encodingthe polypeptides of the present invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences. Alsoincluded in the present invention are nucleic acids encodingpolypeptides of the present invention together with additional,non-coding sequences, including for example, but not limited tonon-coding 5′ and 3′ sequences, vector sequence, sequences used forpurification, probing, or priming. For example, heterologous sequencesinclude transcribed, nontranslated sequences that may play a role in,transcription, and mRNA processing, for example, ribosome binding andstability of mRNA. The heterologous sequences may alternatively compriseadditional coding sequences that provide additional functionalities.Thus, a nucleotide sequence encoding a polypeptide may be fused to a tagsequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the tag amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Chatsworth, Calif.), among others, many of which arecommercially available. For instance, hexa-histidine provides forconvenient purification of the fusion protein (See, Gentz et al., (1989)Proc Natl Acad Sci USA 86:8214). The “HA” tag is another peptide usefulfor purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein (See, Wilson et al., (1984) Cell37:767-78). As discussed above, other such fusion proteins include KRIBcDNA fused to Fc at the N- or C-terminus.

III. Recombinant Vectors of the Invention

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, that is either double-stranded orsingle-stranded, and that comprises at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

The present invention relates to recombinant vectors comprising any oneof the polynucleotides described herein.

The present invention encompasses a family of recombinant vectors thatcomprise polynucleotides encoding KRIB polypeptides of the invention.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide in a suitable cell host,this polynucleotide being amplified every time that the recombinantvector replicates. The inserted polynucleotide can be one that encodesKRIB polypeptides of the invention.

A second preferred embodiment of the recombinant vectors according tothe invention consists of expression vectors comprising polynucleotidesencoding KRIB polypeptides of the invention. Within certain embodiments,expression vectors are employed to express a KRIB polypeptide of theinvention, preferably a modified KRIB described in the presentinvention, which can be then purified and, for example, be used as atreatment for metabolic disorders, or simply to reduce body mass ofindividuals.

Expression requires that appropriate signals are provided in thevectors, said signals including various regulatory elements, such asenhancers/promoters from both viral and mammalian sources, that driveexpression of the genes of interest in host cells. Dominant drugselection markers for establishing permanent, stable, cell clonesexpressing the products are generally included in the expression vectorsof the invention, as they are elements that link expression of the drugselection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding a KRIB polypeptide of theinvention, or a modified KRIB as described-herein, or variants orfragments thereof, under the control of a regulatory sequence selectedamong KRIB polypeptides, or alternatively under the control of anexogenous regulatory sequence.

Consequently, preferred expression vectors of the invention are selectedfrom the group consisting of: (a) a KRIB regulatory sequence and drivingthe expression of a coding polynucleotide operably linked thereto; and(b) a KRIB coding sequence of the invention, operably linked toregulatory sequences allowing its expression in a suitable cell hostand/or host organism.

In a preferred embodiment of the invention, said expression vector is agene therapy vector.

Some of the elements that can be found in the vectors of the presentinvention are described in further detail in the following sections.

1) General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, oreven a linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

-   -   (1) a genetic element or elements having a regulatory role in        gene expression, for example promoters or enhancers. Enhancers        are cis-acting elements of DNA, usually from about 10 to 300 bp        in length that act on the promoter to increase the        transcription;    -   (2) a structural or coding sequence which is transcribed into        mRNA and eventually translated into a polypeptide, said        structural or coding sequence being operably linked to the        regulatory elements described in (1); and    -   (3) appropriate transcription initiation and termination        sequences. Structural units intended for use in yeast or        eukaryotic expression systems preferably include a leader        sequence enabling extracellular secretion of translated protein        by a host cell. Alternatively, when a recombinant protein is        expressed without a leader or transport sequence, it may include        a N-terminal residue. This residue may or may not be        subsequently cleaved from the expressed recombinant protein to        provide a final product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsites, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

2) Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors of thepresent invention are chosen taking into account the cell host in whichthe heterologous gene is expressed. The particular promoter employed tocontrol the expression of a nucleic acid sequence of interest is notbelieved to be important, so long as it is capable of directing theexpression of the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell, such as, for example, a human or aviral promoter.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Preferred bacterial promoters are the LacI,LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt,lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter,or the p10 protein promoter from baculovirus (Kit Novagen) (Smith etal., (1983) Mol Cell Biol 3:2156-65; O'Reilly et al., 1992), the lambdaPR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.In addition, promoters specific for a particular cell type may bechosen, such as those facilitating expression in adipose tissue, muscletissue, or liver. Selection of a convenient vector and promoter is wellwithin the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic engineering. For example, one may refer toSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), or also to theprocedures described by Fuller et al. (1996) Immunology in CurrentProtocols in Molecular Biology.

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

Vectors containing the appropriate DNA sequence as described above canbe utilized to transform an appropriate host to allow the expression ofthe desired polypeptide or polynucleotide.

3) Selectable Markers

Such markers would confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

4) Preferred Vectors

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), andpGEM1 (Promega Biotec, Madison, Wis., USA).

Large numbers of other suitable vectors are known to those of skill inthe art, and are commercially available, such as the following bacterialvectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT,pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia);pQE-30 (QIAexpress).

Baculovirus Vectors

A suitable vector for the expression of polypeptides of the invention isa baculovirus vector that can be propagated in insect cells and ininsect cell lines. A specific suitable host vector system is thepVL1392/1393 baculovirus transfer vector (Pharmingen) that is used totransfect the SF9 cell line (ATCC No CRL 1711) which is derived fromSpodoptera frugiperda.

Other suitable vectors for the expression of a KRIB polypeptide in abaculovirus expression system include those described by Chai et al.(1993; Biotechnol Appl Biochem 18 (Pt 3):259-73); Vlasak et al. (1983;Eur J Biochem 135:123-6); and Lenhard et al. (1996; Gene 169:187-90).

Plasmid Vectors

A suitable vector for the expression of polypeptides of the invention isa plasmid vector that contains an SV40-derived origin of replication andthat can be used for transient transfection of COS cells (ATCC NoCRL1650; No CRL1651). Plasmid vectors suitable for transienttransfection of COS cells include but are not limited to CDM8(Invitrogen).

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996; Semin Interv Cardiol 1:203-8) orOhno et al. (1994; Science 265:781-4). Another preferred recombinantadenovirus according to this specific embodiment of the presentinvention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or anadenovirus of animal origin (French patent application No. FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vivo gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA86:9079-83), Julan et al., (1992) J. Gen. Virol 3:3251-3255 and Neda etal., ((1991) J Biol Chem 266:14143-6).

Yet another viral vector system that is contemplated by the inventionconsists of the adeno-associated virus (AAV). The adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., (1992) CurrTop Microbiol Immunol 158:97-129). It is also one of the few virusesthat may integrate its DNA into non dividing cells, and exhibits a highfrequency of stable integration (Flotte et al., (1992) Am J Respir CellMol Biol 7:349-56; Samulski et al., (1989) J Virol 63:3822-8; McLaughlinet al., (1989) Am J Hum Genet 59:561-569). One advantageous feature ofAAV derives from its reduced efficacy for transducing primary cellsrelative to transformed cells.

5) Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides of the invention,these constructs must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transforming celllines, or in vivo or ex vivo, as in the treatment of certain disorderstates.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calcium phosphate precipitation(Graham et al., (1973) Virology 54:536-9; Chen et al., (1987) Mol CellBiol 7:2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol 5:1188-90),electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol 6:716-8; Potteret al., (1984) Proc Natl Acad Sci USA 81:7161-5.), direct microinjection(Harland et al., (1985) J Cell Biol 101:1094-9), DNA-loaded liposomes(Nicolau et al., (1982) Biochim Biophys Acta 721:185-90; Fraley et al.,(1979) Proc Natl Acad Sci USA 76:3348-52), and receptor-mediatedtransfection (Wu and Wu, (1987) J Biol Chem 262:4429-32; Wu and Wu(1988) Biochemistry 27:887-92). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No. WO 95/11307 (Institut Pasteur,INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.(1996) Nature Medicine 2:888-892 and of Huygen et al. ((1996) Nat Med2:893-8).

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.((1990) Curr Genet 17:97-103).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987)Methods Enzymol. 149:157-76). These liposomes may further be targeted tocells expressing LSR by incorporating leptin, triglycerides, or otherknown LSR ligands into the liposome membrane.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired KRIBpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

IV. Recombinant Cells of the Invention

Another object of the invention consists of host cells recombinant for,i.e., that have been transformed or transfected with one of thepolynucleotides described herein, and more precisely a polynucleotidecomprising a polynucleotide encoding a KRIB polypeptide of the inventionsuch as any one of those described in “Polynucleotides of theInvention”. These polynucleotides can be present in cells as a result oftransient or stable transfection. The invention includes host cells thatare transformed (prokaryotic cells) or that are transfected (eukaryoticcells) with a recombinant vector such as any one of those described in“Recombinant Vectors of the Invention”.

Generally, a recombinant host cell of the invention comprises at leastone of the polynucleotides or the recombinant vectors of the inventionthat are described herein.

Preferred host cells used as recipients for the recombinant vectors ofthe invention are the following:

-   -   a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-α        strain), Bacillus subtilis, Salmonella typhimurium, and strains        from species like Pseudomonas, Streptomyces and Staphylococcus,        and    -   b) Eukaryotic host cells: HeLa cells (ATCC No CCL2; No CCL2.1;        No CCL2.2), Cv 1 cells (ATCC No CCL70), COS cells (ATCC No        CRL1650; No CRL1651), Sf-9 cells (ATCC No CRL1711), C127 cells        (ATCC No CRL-1804), 3T3 (ATCC No CRL6361), CHO (ATCC No CCL-61),        human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No        84100501; No 84111301), PLC cells, HepG2, and Hep3B.

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

In a preferred embodiment, recombinant protein expressed by cells thathave been stably or transiently transfected with a recombinant vectorsuch as any one of those described in “Recombinant Vectors of theInvention” is recovered from culture supernatant.

Alternatively, cells may be harvested (typically by centrifugation),disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

Further, according to the invention, these recombinant cells can becreated in vitro or in vivo in an animal, preferably a mammal, mostpreferably selected from the group consisting of mice, rats, dogs, pigs,sheep, cattle, and primates, not to include humans. Recombinant cellscreated in vitro can also be later surgically implanted in an animal,for example. Methods to create recombinant cells in vivo in animals arewell-known in the art.

The present invention also encompasses primary, secondary, andimmortalized homologously recombinant host cells of vertebrate origin,preferably mammalian origin and particularly human origin, that havebeen engineered to: a) insert exogenous (heterologous) polynucleotidesinto the endogenous chromosomal DNA of a targeted gene, b) deleteendogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNAwith exogenous polynucleotides. Insertions, deletions, and/orreplacements of polynucleotide sequences may be to the coding sequencesof the targeted gene and/or to regulatory regions, such as promoter andenhancer sequences, operably associated with the targeted gene.

The present invention further relates to a method of making ahomologously recombinant host cell in vitro or in vivo, wherein theexpression of a targeted gene not normally expressed in the cell isaltered. Preferably the alteration causes expression of the targetedgene under normal growth conditions or under conditions suitable forproducing the polypeptide encoded by the targeted gene. The methodcomprises the steps of: (a) transfecting the cell in vitro or in vivowith a polynucleotide construct, the polynucleotide constructcomprising; (i) a targeting sequence; (ii) a regulatory sequence and/ora coding sequence; and (iii) an unpaired splice donor site, ifnecessary, thereby producing a transfected cell; and (b) maintaining thetransfected cell in vitro or in vivo under conditions appropriate forhomologous recombination.

The present invention further relates to a method of altering theexpression of a targeted gene in a cell in vitro or in vivo wherein thegene is not normally expressed in the cell, comprising the steps of: (a)transfecting the cell in vitro or in vivo with a polynucleotideconstruct, the polynucleotide construct comprising: (i) a targetingsequence; (ii) a regulatory sequence and/or a coding sequence; and (iii)an unpaired splice donor site, if necessary, thereby producing atransfected cell; and (b) maintaining the transfected cell in vitro orin vivo under conditions appropriate for homologous recombination,thereby producing a homologously recombinant cell; and (c) maintainingthe homologously recombinant cell in vitro or in vivo under conditionsappropriate for expression of the gene.

The present invention further relates to a method of making apolypeptide of the present invention by altering the expression of atargeted endogenous gene in a cell in vitro or in vivo wherein the geneis not normally expressed in the cell, comprising the steps of: a)transfecting the cell in vitro with a polynucleotide construct, thepolynucleotide construct comprising: (i) a targeting sequence; (ii) aregulatory sequence and/or a coding sequence; and (iii) an unpairedsplice donor site, if necessary, thereby producing a transfected cell;(b) maintaining the transfected cell in vitro or in vivo underconditions appropriate for homologous recombination, thereby producing ahomologously recombinant cell; and c) maintaining the homologouslyrecombinant cell in vitro or in vivo under conditions appropriate forexpression of the gene thereby making the polypeptide.

The present invention further relates to a polynucleotide construct thatalters the expression of a targeted gene in a cell type in which thegene is not normally expressed. This occurs when a polynucleotideconstruct is inserted into the chromosomal DNA of the target cell,wherein the polynucleotide construct comprises: a) a targeting sequence;b) a regulatory sequence and/or coding sequence; and c) an unpairedsplice-donor site, if necessary. Further included are polynucleotideconstructs, as described above, wherein the construct further comprisesa polynucleotide that encodes a polypeptide and is in-frame with thetargeted endogenous gene after homologous recombination with chromosomalDNA.

The compositions may be produced, and methods performed, by techniquesknown in the art, such as those described in U.S. Pat. Nos. 6,054,288;6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734;International Publication Nos: WO96/29411, WO 94/12650; and scientificarticles described by Koller et al., (1994) Annu Rev Immunol 10:705-730;the disclosures of each of which are incorporated by reference in theirentireties).

The expression of KRIB in mammalian, and typically human, cells may berendered defective, or alternatively it may be enhanced, with theinsertion of a KRIB genomic or cDNA sequence with the replacement of theKRIB genes counterpart in the genome of an animal cell by a KRIBpolynucleotide according to the invention. These genetic alterations maybe generated by homologous recombination events using specific DNAconstructs that have been previously described.

One kind of host cell that may be used are mammalian zygotes, such asmurine zygotes. For example, murine zygotes may undergo microinjectionwith a purified DNA molecule of interest, for example a purified DNAmolecule that has previously been adjusted to a concentration range from1 ng/ml—for BAC inserts-3 ng/μl—for P1 bacteriophage inserts—in 10 mMTris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine,and 70 μM spermidine. When the DNA to be microinjected has a large size,polyamines and high salt concentrations can be used in order to avoidmechanical breakage of this DNA, as described by Schedl et al ((1993)Nature 362:258-61).

Any one of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells which provide the appropriatesignals to preserve this embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells are primary embryonicfibroblasts that are established from tissue of day 13-day 14 embryos ofvirtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (1993; Methods Enzymol 225:803-23) andare inhibited in growth by irradiation, such as described by Robertson((1987) Embryo-derived stem cell lines. In: E. J. Robertson Ed.Teratocarcinomas and embrionic stem cells: a practical approach. IRLPress, Oxford), or by the presence of an inhibitory concentration ofLIF, such as described by Pease and Williams (1990; Exp Cell Res190:209-11).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

V. DNA Construct that Enables Directing Temporal and Spatial KRIB GeneExpression in Transgenic Animals

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the KRIB genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycine resistance (two); and (c) asecond nucleotide sequence that is comprised in the KRIB genomicsequence, and is located on the genome downstream the first KRIBnucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene (Thomas etal., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprtgene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteriatoxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990),which disclosures are hereby incorporated by reference in theirentireties. Preferably, the positive selection marker is located withina KRIB exon sequence so as to interrupt the sequence encoding a KRIBprotein. These replacement vectors are described, for example, by Thomaset al. (1986; 1987), Mansour et al. (1988) and Koller et al. (1992).

The first and second nucleotide sequences (a) and (c) may beindifferently located within a KRIB regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.Preferably the nucleotide sequences (a) and (c) comprise all or part ofodd SEQ ID NOs: 1-9.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System

These new DNA constructs make use of the site specific recombinationsystem of the P1 phage. The P1 phage possesses a recombinase called Crethat interacts specifically with a 34 base pairs loxP site. The loxPsite is composed of two palindromic sequences of 13 bp separated by a 8bp conserved sequence (Hoess et al., 1986), which disclosure is herebyincorporated by reference in its entirety. The recombination by the Creenzyme between two loxP sites having an identical orientation leads tothe deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu et al. (1993, 1994), whichdisclosures are hereby incorporated by reference in their entireties.Briefly, a nucleotide sequence of interest to be inserted in a targetedlocation of the genome harbors at least two loxP sites in the sameorientation and located at the respective ends, of a nucleotide sequenceto be excised from the recombinant genome. The excision event requiresthe presence of the recombinase (Cre) enzyme within the nucleus of therecombinant cell host. The recombinase enzyme may be brought at thedesired time either by (a) incubating the recombinant cell hosts in aculture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as described by Araki et al.(1995), which disclosure is hereby incorporated by reference in itsentirety, or by lipofection of the enzyme into the cells, such asdescribed by Baubonis et al. (1993), which disclosure, is herebyincorporated by reference in its entirety; (b) transfecting the cellhost with a vector comprising the Cre coding sequence operably linked toa promoter functional in the recombinant cell host, which promoter beingoptionally inducible, said vector being introduced in the recombinantcell host, such as described by Gu et al. (1993) and Sauer et al.(1988), which disclosures are hereby incorporated by reference in theirentireties; (c) introducing in the genome of the cell host apolynucleotide comprising the Cre coding sequence operably linked to apromoter functional in the recombinant cell host, which promoter isoptionally inducible, and said polynucleotide being inserted in thegenome of the cell host either by a random insertion event or anhomologous recombination event, such as described by Gu et al. (1994).

In a specific embodiment, the vector containing the sequence to beinserted in the KRIB genes by homologous recombination is constructed insuch a way that selectable markers are flanked by loxP sites of the sameorientation, it is possible, by treatment by the Cre enzyme, toeliminate the selectable markers while leaving the KRIB sequences ofinterest that have been inserted by an homologous recombination event.Again, two selectable markers are needed: a positive selection marker toselect for the recombination event and a negative selection marker toselect for the homologous recombination event. Vectors and methods usingthe Cre-loxP system are described by Zou et al. (1994), which disclosureis hereby incorporated by reference in its entirety.

Thus, a second preferred DNA construct of the invention comprises, from5′-end to 3′-end: (a) a first nucleotide sequence that is comprised inthe KRIB genomic sequence; (b) a nucleotide sequence comprising apolynucleotide encoding a positive selection marker, said nucleotidesequence comprising additionally two sequences defining a siterecognized by a recombinase, such as a loxP site, the two sites beingplaced in the same orientation; and (c) a second nucleotide sequencethat is comprised in the KRIB genomic sequence, and is located on thegenome downstream of the first KRIB nucleotide sequence (a). Preferablythe nucleotide sequences (a) and (c) comprise all or part of odd SEQ IDNOs: 1-9.

The sequences defining a site recognized by a recombinase, such as aloxP site, are preferably located within the nucleotide sequence (b) atsuitable locations bordering the nucleotide sequence for which theconditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA constructdescribed above, the excision of the polynucleotide fragment bordered bythe two sites recognized by a recombinase, preferably two loxP sites, isperformed at a desired time, due to the presence within the genome ofthe recombinant host cell of a sequence encoding the Cre enzyme operablylinked to a promoter sequence, preferably an inducible promoter, morepreferably a tissue-specific promoter sequence and most preferably apromoter sequence which is both inducible and tissue-specific, such asdescribed by Gu et al. (1994).

The presence of the Cre enzyme within the genome of the recombinant cellhost may result from the breeding of two transgenic animals, the firsttransgenic animal bearing the KRIB-derived sequence of interestcontaining the loxP sites as described above and the second transgenicanimal bearing the Cre coding sequence operably linked to a suitablepromoter sequence, such as described by Gu et al. (1994).

Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton and Graham (1995)and Kanegae et al. (1995), which disclosures are hereby incorporated byreference in their entireties.

The DNA constructs described above may be used to introduce a desirednucleotide sequence of the invention, preferably a KRIB genomic sequenceor a KRIB cDNA sequence, and most preferably an altered copy of a KRIBgenomic or cDNA sequence, within a predetermined location of thetargeted genome, leading either to the generation of an altered copy ofa targeted gene (knock-out homologous recombination) or to thereplacement of a copy of the targeted gene by another copy sufficientlyhomologous to allow an homologous recombination event to occur (knock-inhomologous recombination).

VI. Transgenic Animals

The present invention also provides methods and compositions for thegeneration of non-human animals and plants that express the recombinantKRIB polypeptides, of the present invention. The animals or plants canbe transgenic, i.e. each of their cells contains a gene encoding a KRIBpolypeptide, or, alternatively, a polynucleotide encoding a KRIBpolypeptide can be introduced into somatic cells of the animal or plant,e.g. into mammary secretory epithelial cells of a mammal. In preferredembodiments, the non-human animal is a mammal such as a cow, sheep,goat, pig, or rabbit.

Methods of making transgenic animals such as mammals are well known tothose of skill in the art, and any such method can be used in thepresent invention. Briefly, transgenic mammals can be produced, e.g., bytransfecting a pluripotential stem cell such as an ES cell with apolynucleotide encoding a polypeptide of interest. Successfullytransformed ES cells can then be introduced into an early stage embryothat is then implanted into the uterus of a mammal of the same species.In certain cases, the transformed (“transgenic”) cells will comprisepart of the germ line of the resulting animal, and adult animalscomprising the transgenic cells in the germ line can then be mated toother animals, thereby eventually producing a population of transgenicanimals that have the transgene in each of their cells, and which canstably transmit the transgene to each of their offspring. Other methodsof introducing the polynucleotide can be used, for example introducingthe polynucleotide encoding the polypeptide of interest into afertilized egg or early stage embryo via microinjection. Alternatively,the transgene may be introduced into an animal by infection of zygoteswith a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.Natl. Acad. Sci. USA 73, 1260-1264). Methods of making transgenicmammals are described, e.g., in Wall et al. (1992) J Cell Biochem 199249:113-20; Hogan, et al. (1986) in Manipulating the mouse embryo. ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; in WO 91/08216, or in U.S. Pat. No. 4,736,866.

In a preferred method, the polynucleotides are microinjected into thefertilized oocyte. Typically, fertilized oocytes are microinjected usingstandard techniques, and then cultured in vitrountil a “pre-implantationembryo” is obtained. Such pre-implantation embryos preferably containapproximately 16 to 150 cells. Methods for culturing fertilized oocytesto the pre-implantation stage are described, e.g., by Gordon et al.((1984) Methods in Enzymology, 101, 414); Hogan et al. ((1986) inManipulating the mouse embryo. A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y) (for the mouse embryo);Hammer et al. ((1985) Nature, 315, 680) (for rabbit and porcineembryos); Gandolfi et al. ((1987) J. Reprod. Fert. 81, 23-28); Rexroadet al. ((1988) J. Anim. Sci. 66, 947-953) (for ovine embryos); andEyestone et al. ((1989) J. Reprod. Fert. 85, 715-720); Camous et al.((1984) J. Reprod. Fert. 72, 779-785); and Heyman et al. ((1987)Theriogenology 27, 5968) (for bovine embryos); the disclosures of eachof which are incorporated herein in their entireties. Pre-implantationembryos are then transferred to an appropriate female by standardmethods to permit the birth of a transgenic or chimeric animal,depending upon the stage of development when the transgene isintroduced.

As the frequency of transgene incorporation is often low, the detectionof transgene integration in pre-implantation embryos is often desirableusing any of the herein-described methods. Any of a number of methodscan be used to detect the presence of a transgene in a pre-implantationembryo. For example, one or more cells may be removed from thepre-implantation embryo, and the presence or absence of the transgene inthe removed cell or cells can be detected using any standard method e.g.PCR. Alternatively, the presence of a transgene can be detected in uteroor post partum using standard methods.

In a particularly preferred embodiment of the present invention,transgenic mammals are generated that secrete recombinant KRIBpolypeptides in their milk. As the mammary gland is a highly efficientprotein-producing organ, such methods can be used to produce proteinconcentrations in the gram per liter range, and often significantlymore. Preferably, expression in the mammary gland is accomplished byoperably linking the polynucleotide encoding the KRIB polypeptide to amammary gland specific promoter and, optionally, other regulatoryelements. Suitable promoters and other elements include, but are notlimited to, those derived from mammalian short and long WAP, alpha,beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes,as well as the the mouse mammary tumor virus (MMTV) promoter. Suchpromoters and other elements may be derived from any mammal, including,but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guineapigs. Promoter and other regulatory sequences, vectors, and otherrelevant teachings are provided, e.g., by Clark (1998) J Mammary GlandBiol Neoplasia 3:337-50; Jost et al. (1999) Nat Biotechnol 17:1604; U.S.Pat. Nos. 5,994,616; 6,140,552; 6,013,857; Sohn et al. (1999) DNA CellBiol. 18:845-52; Kim et al. (1999) J Biochem (Japan) 126:320-5; Soulieret al. (1999) Euro J Biochem 260:533-9; Zhang et al. (1997) Chin JBiotech 13:271-6; Rijnkels et al. (1998) Transgen Res 7:5-14; Korhonenet al. (1997) Euro J Biochem 245:482-9; Uusi-Oukari et al. (1997)Transgen Res 6:75-84; Hitchin et al. (1996) Prot Expr Purif 7:247-52;Platenburg et al. (1994) Transgen Res 3:99-108; Heng-Cherl et al. (1993)Animal Biotech. 4:89-107; and Christa et al. (2000) Euro J Biochem267:1665-71; the entire disclosures of each of which is hereinincorporated by reference.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens etal. (2000) Biochim Biophys. Acta 1523:161-171), in association with aligand that can lead to receptor-mediated endocytosis of the construct(see, e.g., Sobolev et al. (1998) 273:7928-33), or in a viral vectorsuch as a retroviral vector, e.g. the Gibbon ape leukemia virus (see,e.g., Archer et al. (1994) PNAS 91:6840-6844). In any of theseembodiments, the polynucleotide may be operably linked to a mammarygland specific promoter, as described above, or, alternatively, anystrongly expressing promoter such as CMV or MoMLV LTR.

The suitability of any vector, promoter, regulatory element, etc. foruse in the present invention can be assessed beforehand by transfectingcells such as mammary epithelial cells, e.g. MacT cells (bovine mammaryepithelial cells) or GME cells (goat mammary epithelial cells), in vitroand assessing the efficiency of transfection and expression of thetransgene in the cells.

For in vivo administration, the polynucleotides can be administered inany suitable formulation, at any of a range of concentrations (e.g.1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g. 1-100 ml,preferably 1 to 20 ml), and can be administered any number of times(e.g. 1, 2, 3, 5, or 10 times), at any frequency (e.g. every 1, 2, 3, 5,10, or any number of days). Suitable concentrations, frequencies, modesof administration, etc. will depend upon the particular polynucleotide,vector, animal, etc., and can readily be determined by one of skill inthe art.

In a preferred embodiment, a retroviral vector such as as Gibbon apeleukemia viral vector is used, as described in Archer et al. ((1994)PNAS 91:6840-6844). As retroviral infection typically requires celldivision, cell division in the mammary glands can be stimulated inconjunction with the administration of the vector, e.g. using a factorsuch as estrodiol benzoate, progesterone, reserpine, or dexamethasone.Further, retroviral and other methods of infection can be facilitatedusing accessory compounds such as polybrene.

In any of the herein-described methods for obtaining KRIB polypeptidesfrom milk, the quantity of milk obtained, and thus the quantity of KRIBpolypeptides produced, can be enhanced using any standard method oflactation induction, e.g. using hexestrol, estrogen, and/orprogesterone.

The polynucleotides used in such embodiments can either encode afull-length KRIB polypeptide or a KRIB polypeptide fragment. Typically,the encoded polypeptide will include a signal sequence to ensure thesecretion of the protein into the milk. Where a full length KRIBsequence is used, the full-length protein can, e.g., be isolated frommilk and cleaved in vitro using a suitable protease. Alternatively, asecond, protease-encoding polynucleotide can be introduced into theanimal or into the mammary gland cells, whereby expression of theprotease results in the cleavage of the KRIB polypeptide in vivo,thereby allowing the direct isolation of KRIB fragments from milk.

VII. Pharmaceutical or Physiologically Acceptable Compositions of theInvention

The KRIB polypeptides of the invention can be administered to non-humananimals and/or humans, alone or in pharmaceutical or physiologicallyacceptable compositions where they are mixed with suitable carriers orexcipient(s). The pharmaceutical or physiologically acceptablecomposition is then provided at a therapeutically effective dose. Atherapeutically effective dose refers to that amount of a KRIBpolypeptide sufficient to result in prevention or amelioration ofsymptoms or physiological status of metabolic disorders as determined bythe methods described herein. A therapeutically effective dose can alsorefer to the amount of a KRIB polypeptide necessary for a reduction inweight or a prevention of an increase in weight or prevention of anincrease in the rate of weight gain in persons desiring this affect forcosmetic reasons. A therapeutically effective dosage of a KRIBpolypeptide of the invention is that dosage that is adequate to promoteweight loss or weight gain with continued periodic use oradministration. Techniques for formulation and administration of KRIBpolypeptides may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition.

Other disorders that KRIB polypeptides of the invention could be used totreat or prevent include, but are not limited to, obesity andobesity-related disorders such as obesity, impaired glucose tolerance,insulin resistance, atherosclerosis, atheromatous disorder, heartdisorder, hypertension, stroke, Syndrome X, Non-Insulin DependentDiabetes Mellitus (NIDDM, or Type II diabetes) and Insulin DependentDiabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, renallesions. Heart disorder includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other obesity-relateddisorders of the invention include cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia. The KRIBpolypeptides may also be used to enhance physical performance duringwork or exercise or enhance a feeling of general well-being. Physicalperformance activities include walking, running, jumping, lifting and/orclimbing.

The KRIB polypeptides or antagonists thereof may also be used to treatdyslexia, attention-deficit disorder (ADD),attention-deficit/hyperactivity disorder (ADHD), and psychiatricdisorders such as schizophrenia by modulating fatty acid metabolism,more specifically, the production of certain long-chain polyunsaturatedfatty acids.

It is expressly considered that the KRIB polypeptides of the inventionmay be provided alone or in combination with other pharmaceutically orphysiologically acceptable compounds. Other compounds useful for thetreatment of obesity and other disorders are currently well-known in theart.

In a preferred embodiment, the KRIB polypeptides are useful for, andused in, the treatment of insulin resistance and diabetes using methodsdescribed herein and known in the art. More particularly, a preferredembodiments relates to process for the therapeutic modification andregulation of glucose metabolism in an animal or human subject, whichcomprises administering to a subject in need of treatment (alternativelyon a timed daily basis) KRIB polypeptide (or polynucleotide encodingsaid polypeptide) in dosage amount and for a period sufficient to reduceplasma glucose levels in said animal or human subject.

Further preferred embodiments relate to methods for the prophylaxis ortreatment of diabetes comprising administering to a subject in need oftreatment (alternatively on a timed daily basis) a KRIB polypeptide (orpolynucleotide encoding said polypeptide) in dosage amount and for aperiod sufficient to reduce plasma glucose levels in said animal orhuman subject.

Routes of Administration.

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. A particularly useful methodof administering compounds for promoting weight loss involves surgicalimplantation, for example into the abdominal cavity of the recipient, ofa device for delivering KRIB polypeptides over an extended period oftime. Other particularly preferred routes of administration are aerosoland depot formulation. Sustained release formulations, particularlydepot, of the invented medicaments are expressly contemplated.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable acceptable carrier and atleast one polypeptide that is a KRIB polypeptide of the invention. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer suchas a phosphate or bicarbonate buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to increase leptin orlipoprotein uptake or binding in an in vitro system. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50, (the dose lethal to 50% of the testpopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD50and ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintain orprevent weight loss or gain, depending on the particular situation.Dosages necessary to achieve these effects will depend on individualcharacteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-90%; andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

A preferred dosage range for the amount of a KRIB polypeptide of theinvention, which can be administered on a daily or regular basis toachieve desired results, including a reduction in levels of circulatingplasma triglyceride-rich lipoproteins, range from 0.05-1.0 mg/kg bodymass. A more preferred dosage range is from 0.1-5 mg/kg. A morepreferred dose is 0.25-2.5 mg/kg. Of course, these daily dosages can bedelivered or administered in small amounts periodically during thecourse of a day. It is noted that these dosage ranges are only preferredranges and are not meant to be limiting to the invention.

VIII. Methods of Treatment

The invention is drawn inter alia to methods of preventing or treatingmetabolic disorders comprising providing an individual in need of suchtreatment with a KRIB polypeptide of the invention. Preferably, the KRIBpolypeptide has metabolic activity either in vitro or in vivo.Preferably the KRIB polypeptide is provided to the individual in apharmaceutical composition that is preferably taken orally. Preferablythe individual is a mammal, and most preferably a human. In preferredembodiments, the metabolic disorder is selected from the groupconsisting of atherosclerosis, cardiovascular disorder, impaired glucosetolerance, insulin resistance, hypertension, stroke, Syndrome X, Type Idiabetes, Type II diabetes and lipoatrophic diabetes. Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy andrenal lesions. Heart disorder includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic disorders to be treated by compounds of the invention includehyperlipidemia, hypertriglyceridemia, and hyperuricemia. Yet othermetabolic disorders of the invention include cachexia, wasting,AIDS-related weight loss, cancer-related weight loss, neoplasia-relatedweight loss, anorexia, and bulimia. In preferred embodiments, KRIBpolypeptides in pharmaceutical compositions are used to modulate bodyweight in healthy individuals for cosmetic reasons.

The invention also features a method of preventing or treating metabolicdisorders comprising providing an individual in need of such treatmentwith a compound identified by assays of the invention (described inSection VI of the Preferred Embodiments of the Invention and in theExamples). Preferably these compounds antagonize or agonize effects ofKRIB polypeptides in cells in vitro, muscles ex vivo, or in animalmodels. Alternatively, these compounds agonize or antagonize the effectsof KRIB polypeptides on leptin and/or lipoprotein uptake and/or binding.Optionally, these compounds prevent the interaction, binding, or uptakeof KRIB polypeptides with LSR in vitro or in vivo. Preferably, thecompound is provided to the individual in a pharmaceutical compositionthat is preferably taken orally. Preferably the individual is a mammal,and most preferably a human. In preferred embodiments, the metabolicdisorder is selected from the group consisting of obesity and metabolicdisorders such as atherosclerosis, heart disorder, insulin resistance,hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes, andlipoatrophic diabetes. Diabetes-related complications to be treated bythe methods of the invention include microangiopathic lesions, ocularlesions, retinopathy, neuropathy and renal lesions. Heart disorderincludes, but is not limited to, cardiac insufficiency, coronaryinsufficiency, and high blood pressure. Other metabolic disorders to betreated by compounds of the invention include hyperlipidemia,hypertriglyceridemia, and hyperuricemia.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance,alone, without combination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type II diabetes or insulin resistance, alone, without combinationof insulin therapy. In still a further preferred embodiment, the controlof body weight is due in part or in whole to a decrease in mass of 1)subcutaneous adipose tissue and/or 2) visceral (omental) adipose tissue.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method of preventing weight gain in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance oralternatively for cosmetic reasons.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method of reducing weight in some individuals, particularly those withType I diabetes, Type II diabetes, or insulin resistance oralternatively for cosmetic reasons.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method of maintaining weight loss in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance oralternatively for cosmetic reasons.

In a further preferred embodiment, the present invention may be used incomplementary therapy, particularly in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, toimprove their weight or glucose control in combination with an insulinsecretagogue or an insulin sensitising agent. Preferably, the insulinsecretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate(BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide,chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.Preferably, the insulin sensitising agent is selected from metformin,ciglitazone, troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of some individuals, particularly those withType I diabetes, Type II diabetes, or insulin resistance, alone, withoutan insulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an oral insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin some individuals, particularly those with Type I diabetes, Type IIdiabetes, or insulin resistance.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type I diabetes, Type II diabetes, or insulinresistance, in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type II diabetes or insulin resistance, withoutinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an inhibitor of the progression fromimpaired glucose tolerance to insulin resistance.

IX. Ligands Interacting with KRIB Polypeptides

For the purpose of the present invention, a KRIB ligand means amolecule, such as a protein, a peptide, an antibody or any syntheticchemical compound capable of binding to a KRIB protein or one of itsfragments or variants.

In the Ligand screening method according to the present invention, abiological sample or a defined molecule to be tested as a putativeLigand of a KRIB protein is brought into contact with the correspondingpurified KRIB protein, for example the corresponding purifiedrecombinant KRIB protein produced by a recombinant cell host asdescribed herein, in order to form a complex between this protein andthe putative Ligand molecule to be tested.

As an illustrative example, to study the interaction of a KRIB protein,or a fragment comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selectedfrom the group consisting of even SEQ ID NOs: 2-10, with drugs or smallmolecules, such as molecules generated through combinatorial chemistryapproaches, the microdialysis coupled to HPLC method described by Wanget al. (1997) or the affinity capillary electrophoresis method describedby Bush et al. (1997), the disclosures of which are incorporated byreference, can be used.

In further methods, peptides, drugs, fatty acids, lipoproteins, or smallmolecules which interact with a KRIB protein, or a fragment comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of a polypeptide selected from the group consisting ofsequences of even SEQ ID NOs: 2-10 may be identified using assays suchas the following. The molecule to be tested for binding is labelled witha detectable label, such as a fluorescent, radioactive, or enzymatic tagand placed in contact with immobilized KRIB protein, or a fragmentthereof under conditions that permit specific binding to occur. Afterremoval of non-specifically bound molecules, bound molecules aredetected using appropriate means.

Various candidate substances or molecules can be assayed for interactionwith a KRIB polypeptide. These substances or molecules include, withoutbeing limited to, natural or synthetic organic compounds or molecules ofbiological origin such as polypeptides. When the candidate substance ormolecule comprises a polypeptide, this polypeptide may be the resultingexpression product of a phage clone belonging to a phage-based randompeptide library, or alternatively the polypeptide may be the resultingexpression product of a cDNA library cloned in a vector suitable forperforming a two-hybrid screening assay.

A. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with a KRIB protein, or afragment thereof comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selectedfrom the group consisting of sequences of even SEQ ID NOs: 2-10, canalso be found using affinity columns which contain the KRIB protein, ora fragment thereof. The KRIB protein, or a fragment thereof, may beattached to the column using conventional techniques including chemicalcoupling to a suitable column matrix such as agarose, Affi Gel®, orother matrices familiar to those of skill in art. In some embodiments ofthis method, the affinity column contains chimeric proteins in which theKRIB protein, or a fragment thereof, is fused to glutathion Stransferase (GST). A mixture of cellular proteins or pool of expressedproteins as described above is applied to the affinity column. Proteinsor other molecules interacting with the KRIB protein, or a fragmentthereof, attached to the column can then be isolated and analyzed on 2-Delectrophoresis gel as described in Ramunsen et al. (1997), thedisclosure of which is incorporated by reference. Alternatively, theproteins retained on the affinity column can be purified byelectrophoresis-based methods and sequenced. The same method can be usedto isolate antibodies, to screen phage display products, or to screenphage display human antibodies.

B. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with a KRIB protein, or a fragment comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of a polypeptide selected from the group consisting ofsequences of even SEQ ID NOs: 2-10, can also be screened by using anOptical Biosensor as described in Edwards and Leatherbarrow (1997) andalso in Szabo et al. (1995), the disclosures of which are incorporatedby reference. This technique permits the detection of interactionsbetween molecules in real time, without the need of labelled molecules.This technique is based on the surface plasmon resonance (SPR)phenomenon. Briefly, the candidate Ligand molecule to be tested isattached to a surface (such as a carboxymethyl dextran matrix). A lightbeam is directed towards the side of the surface that does not containthe sample to be tested and is reflected by said surface. The SPRphenomenon causes a decrease in the intensity of the reflected lightwith a specific association of angle and wavelength. The binding ofcandidate Ligand molecules cause a change in the refraction index on thesurface, which change is detected as a change in the SPR signal. Forscreening of candidate Ligand molecules or substances that are able tointeract with the KRIB protein, or a fragment thereof, the KRIB protein,or a fragment thereof, is immobilized onto a surface. This surfacecomprises one side of a cell through which flows the candidate moleculeto be assayed. The binding of the candidate molecule on the KRIBprotein, or a fragment thereof, is detected as a change of the SPRsignal. The candidate molecules tested may be proteins, peptides,carbohydrates, lipids, or small molecules generated by combinatorialchemistry. This technique may also be performed by immobilizingeukaryotic or prokaryotic cells or lipid vesicles exhibiting anendogenous or a recombinantly expressed KRIB protein at their surface.

The main advantage of the method is that it allows the determination ofthe association rate between the KRIB protein and molecules interactingwith the KRIB protein. It is thus possible to select specifically Ligandmolecules interacting with the KRIB protein, or a fragment thereof,through strong or conversely weak association constants.

C. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), which disclosure is herebyincorporated by reference in its entirety, and relies upon the fusion ofa bait protein to the DNA binding domain of the yeast Gal4 protein. Thistechnique is also described in the U.S. Pat. No. 5,667,973 and the U.S.Pat. No. 5,283,173, the technical teachings of both patents being hereinincorporated by reference.

The general procedure of library screening by the two-hybrid assay maybe performed as described by Harper et al. (1993) or as described by Choet al. (1998) or also Fromont-Racine et al. (1997), which disclosuresare hereby incorporated by reference in their entireties.

The bait protein or polypeptide comprises, consists essentially of, orconsists of a KRIB polypeptide or a fragment thereof comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of a polypeptide selected from the group consisting ofsequences of even SEQ ID NOs: 2-10.

More precisely, the nucleotide sequence encoding the KRIB polypeptide ora fragment or variant thereof is fused to a polynucleotide encoding theDNA binding domain of the GAL4 protein, the fused nucleotide sequencebeing inserted in a suitable expression vector, for example pAS2 or pM3.

Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein. Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “prey” polypeptides.

A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pGSEC may be used.

Two different yeast strains are also used. As an illustrative butnon-limiting example the two different yeast strains may be thefollowing:

-   -   Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12,        trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS        GAL-1HS3, cyh^(r));    -   Y187, the phenotype of which is (MATa gal4 gal80 his3 trp1-901        ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet⁻), which is the        opposite mating type of Y190.

Briefly, 20 μg of pAS2/KRIB and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine synthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, beta-gal⁺) are then grown on plateslacking histidine, leucine, but containing tryptophan and cycloheximide(10 mg/ml) to select for loss of pAS2/KRIB plasmid but retention ofpACT-cDNA library plasmids. The resulting Y190 strains are mated withY187 strains expressing KRIB or non-related control proteins; such ascyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper etal. (1993) and by Bram et al. (1993), which disclosures are herebyincorporated by reference in their entireties, and screened for betagalactosidase by filter lift assay. Yeast clones that are beta gal−after mating with the control Gal4 fusions are considered falsepositives.

In another embodiment of the two-hybrid method according to theinvention, interaction between the KRIB or a fragment or variant thereofwith cellular proteins may be assessed using the Matchmaker Two HybridSystem 2 (Catalog No. K1604-1, Clontech). As described in the manualaccompanying the kit, the disclosure of which is incorporated herein byreference, nucleic acids encoding the KRIB protein or a portion thereof,are inserted into an expression vector such that they are in frame withDNA encoding the DNA binding domain of the yeast transcriptionalactivator GAL4. A desired cDNA, preferably human cDNA, is inserted intoa second expression vector such that they are in frame with DNA encodingthe activation domain of GAL4. The two expression plasmids aretransformed into yeast and the yeast are plated on selection mediumwhich selects for expression of selectable markers on each of theexpression vectors as well as GAL4 dependent expression of the HIS3gene. Transformants capable of growing on medium lacking histidine arescreened for GAL4 dependent lacZ expression. Those cells that arepositive in both the histidine selection and the lacZ assay containinteraction between KRIB and the protein or peptide encoded by theinitially selected cDNA insert

X. Assays for Identifying Modulators of KRIB Polypeptide Activity

For the purpose of the present invention, a KRIB modulator means amolecule, such as a protein, a peptide, an antibody or any syntheticchemical compound capable of increasing or decreasing the biologicalactivity of a KRIB protein or one of its fragments or variants, orcapable of modulating the expression of the polynucleotide coding forKRIB or a fragment or variant thereof.

The invention features methods of screening for one or more compoundsthat modulate the activity of KRIB in cells, which includes providingpotential compounds to be tested to the cells. Exemplary assays that maybe used are described in the Examples section. To these assays would beadded compounds to be tested for their inhibitory or stimulatoryactivity as compared to the effects of KRIB polypeptides alone. Otherassays in which an effect is observed based on the addition of KRIBpolypeptides can also be used to screen for modulators of KRIBpolypeptide activity or effects of the presence of KRIB polypeptides oncells. The essential step is to apply an unknown compound and then tomonitor an assay for a change from what is seen when only KRIBpolypeptides are applied to the cell. A change is defined as somethingthat is significantly different in the presence of the compound plusKRIB polypeptide compared to KRIB polypeptide alone. In this case,significantly different would be an “increase” or a “decrease” in ameasurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, or 75%.

The term “modulation” as used herein refers to a measurable change in anactivity. Examples include, but are not limited to, lipolysis stimulatedreceptor (LSR) modulation, leptin modulation, lipoprotein modulation,plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.These effects can be in vitro or preferably in vivo. Modulation of anactivity can be either an increase or a decrease in the activity. Thus,LSR activity can be increased or decreased, leptin activity can beincreased or decreased, and lipoprotein activity can be increased ordecreased. Similarly, FFA, TG, glucose levels and weight can beincreased or decreased in vivo. Free Fatty Acid oxidation can beincreased or decreased in vivo or ex vivo.

In vivo experiments may be performed on any test animal. Prefereably,such animals are mammals. In most preferred embodiments, these mammalsare selected from the group consisting of mice, rats, hamsters and apes.Optionnally, these test animals correspond to animal models for any ofthe disorders described herein. Some test animals that may be used forscreening for KRIB modulators in vivo are described in the Examples atthe end of the present specification.

By “LSR” activity is meant expression of LSR on the surface of the cell,or in a particular conformation, as well as its ability to bind, uptake,and degrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor. Exemplaryassays are provided in the Examples. These assay and other comparableassays can be used to determine/identify compounds that modulate KRIBpolypeptide activity. In some cases it may be important to identifycompounds that modulate some but not all of the KRIB polypeptideactivities, although preferably all activities are modified.

The term “increasing” as used herein refers to the ability of a compoundto increase the activity of KRIB polypeptides in some measurable waycompared to the effect of KRIB polypeptides in its absence. As usedherein, the term “KRIB agonist” refers to a compound that increases theactivity of KRIB polypeptides in some measurable way compared to theeffect of KRIB polypeptides in its absence. As a result of the presenceof the compound leptin binding and/or uptake might increase, forexample, as compared to controls in the presence of the KRIB polypeptidealone. Preferably, an increase in activity is at least 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level ofactivity in the presence of the KRIB polypeptide.

Similarly, the term “decreasing” as used herein refers to the ability ofa compound to decrease an activity in some measurable way compared tothe effect of a KRIB polypeptide in its absence. As used herein, theterm “KRIB antagonist” refers to a compound that decreases the activityof KRIB polypeptides in some measurable way compared to the effect ofKRIB polypeptides in its absence. For example, the presence of thecompound decreases the plasma concentrations of FFA, TG, and glucose inmice. Also as a result of the presence of a compound leptin bindingand/or uptake might decrease, for example, as compared to controls inthe presence of the KRIB polypeptides alone. Preferably, an decrease inactivity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,or 75% as compared to the level of activity in the presence of the KRIBpolypeptides alone.

The invention features a method for identifying a potential compound todecrease body mass in individuals in need of decreasing body masscomprising: a) contacting a cell with a KRIB polypeptide and a candidatecompound; b) detecting a result selected from the group consisting ofLSR modulation, leptin modulation, increase in glucose uptake oroxidation, decrease in blood lipid or triglyceride levels, increase inlipoprotein binding, uptake or degradation; FFA oxidation increase; andc) wherein said result identifies said potential compound if said resultdiffers from said result when said cell is contacted with the KRIBpolypeptide alone.

Alternatively, the invention features a method for identifying apotential compound to increase body mass in individuals in need ofincreasing body mass comprising: a) contacting a cell with a KRIBpolypeptide and a candidate compound; b) detecting a result selectedfrom the group consisting of LSR modulation, leptin modulation, decreasein glucose uptake or oxidation, increase in blood lipid or triglyceridelevels, decrease in lipoprotein binding, uptake or degradation; FFAoxidation decrease; and c) wherein said result identifies said potentialcompound if said result differs from said result when said cell iscontacted with the KRIB polypeptide alone.

In still other preferred embodiments, said potential compound isselected from the group consisting of peptides, peptide libraries,non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments,antibodies, small molecules, proteases and protease inhibitors.

The invention also features methods of screening compounds for one ormore agonists or antagonists of KRIB polypeptide activity, wherein saidactivity is selected from but not restricted to lipid partitioning,lipid metabolism, and insulin-like activity. The invention furtherfeatures methods of screening compounds for one or more agonists orantagonists of KRIB polypeptide activity, wherein said activity isselected from but not restricted to prevention of weight gain, weightreduction, and maintenance of weight loss. Preferred said compound isselected from but is not restricted to small molecular weight organic orinorganic compound, protein, peptide, carbohydrate, or lipid.

The invention further features methods of screening compounds for saidagonists or antagonist of KRIB polypeptide activity comprising: a)contacting said KRIB polypeptide with or without said compound; b)detecting a result on the basis of activity, wherein said activity isselected from but not restricted to lipid partitioning, lipidmetabolism, insulin-like activity, prevention of weight gain, weightreduction, and maintenance of weight loss; and c) wherein said resultidentifies said compound as an agonists or antagonist of KRIBpolypeptide activity if said result with compound differs from saidresult without compound. These methods may be performed in vitro or invivo. Exemplary assays that may be used are described in the Examplesbelow.

XII. Epitopes and Antibody Fusions

A preferred embodiment of the present invention is directed toeiptope-bearing polypeptides and epitope-bearing polypeptide fragments.These epitopes may be “antigenic epitopes” or both an “antigenicepitope” and an “immunogenic epitope”. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the polypeptide is the immunogen. On the other hand, a region ofpolypeptide to which an antibody binds is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of immunogenic epitopesof a protein generally is less than the number of antigenic epitopes.See, e.g., Geysen, et al. (1983) Proc Natl Acad Sci USA 81:39984002. Itis particularly noted that although a particular epitope may not beimmunogenic, it is nonetheless useful since antibodies can be made invitro to any epitope.

An epitope can comprise as few as 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 6 such amino acids, and more often at least 8-10such amino acids. In preferred embodiment, antigenic epitopes comprise anumber of amino acids that is any integer between 3 and 50. Fragmentswhich function as epitopes may be produced by any conventional means.See, e.g., Houghten, R. A., Proc Natl Acad Sci USA 82:5131-5135 (1985),further described in U.S. Pat. No. 4,631,211. Methods for determiningthe amino acids which make up an immunogenic epitope include x-raycrystallography, 2-dimensional nuclear magnetic resonance, and epitopemapping, e.g., the Pepscan method described by H. Mario Geysen et al.(1984); Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No.WO 84/03564; and PCT Publication No. WO 84/03506. Another example is thealgorithm of Jameson and Wolf, Comp. Appl. Biosci. 4:181-186 (1988)(said references incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis, for example, may be performed using thecomputer program PROTEAN, using default parameters (Version 4.0 Windows,DNASTAR, Inc., 1228 South Park Street Madison, Wis.).

The epitope-bearing fragments of the present invention preferablycomprise 6 to 50 amino acids (i.e. any integer between 6 and 50,inclusive) of a polypeptide of the present invention. Also, included inthe present invention are antigenic fragments between the integers of 6and the full-length sequence of the sequence listing. All combinationsof sequences between the integers of 6 and the full-length sequence of apolypeptide of the present invention are included. The epitope-bearingfragments may be specified by either the number of contiguous amino acidresidues (as a sub-genus) or by specific N-terminal and C-terminalpositions (as species) as described above for the polypeptide fragmentsof the present invention.

Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies that specifically bind the epitope (See,Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983). The antibodiesare then used in various techniques such as diagnostic and tissue/cellidentification techniques, as described herein, and in purificationmethods.

Similarly, immunogenic epitopes can be used to induce antibodiesaccording to methods well known in the art (See, Sutcliffe et al.,supra; Wilson et al., supra; Chow, M. et al.; (1985) and Bittle, F. J.et al., (1985). A preferred immunogenic epitope includes thepolypeptides of the sequence listing. The immunogenic epitopes may bepresented together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse) if necessary. Immunogenicepitopes comprising as few as 8 to 10 amino acids have been shown to besufficient to raise antibodies capable of binding to, at the very least,linear epitopes in a denatured polypeptide (e.g., in Western blotting.).

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al., 1985). If in vivo immunization is used,animals may be immunized with free peptide; however, anti-peptideantibody titer may be boosted by coupling of the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH) ortetanus toxoid. For instance, peptides containing cysteine residues maybe coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibody,which can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention including, but not limited to,polypeptides comprising an immunogenic or antigenic epitope can be fusedto heterologous polypeptide sequences. For example, the polypeptides ofthe present invention may be fused with the constant region comprisingportions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of theconstant region (CH1, CH2, CH3, any combination thereof including bothentire domains and portions thereof) resulting in chimeric polypeptides.These fusion proteins facilitate purification, and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker etal., 1988). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone (See, e.g., Fountoulakis et al., 1995). Nucleicacids encoding the above epitopes can also be recombined with a gene ofinterest as an epitope tag to aid in detection and purification of theexpressed polypeptide.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe present invention thereby effectively generating agonists andantagonists of the polypeptides. See, for example, U.S. Pat. Nos.5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al.,(1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo,M. M. and Blasco, R., (1998). (Each of these documents are herebyincorporated by reference). In one embodiment, one or more components,motifs, sections, parts, domains, fragments, etc., of codingpolynucleotides of the invention, or the polypeptides encoded therebymay be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) that specifically bind the polypeptides, and morespecifically, the epitopes of the polypeptides of the present invention.The antibodies of the present invention include IgG (including IgG1,IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM,and IgY. As used herein, the term “antibody” (Ab) is meant to includewhole antibodies, including single-chain whole antibodies, and antigenbinding fragments thereof. In a preferred embodiment the antibodies arehuman antigen binding antibody fragments of the present inventioninclude, but are not limited to, Fab, Fab′F(ab)2 and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies, which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodiesthat are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,and trispecific or have greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991);U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;Kostelny, S. A. et al. (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or epitope-bearing portion(s) of a polypeptideof the present invention, which are recognized or specifically bound bythe antibody. In the case of proteins of the present invention secretedproteins, the antibodies may specifically bind a full-length proteinencoded by a nucleic acid of the present invention, a mature protein(i.e., the protein generated by cleavage of the signal peptide) encodedby a nucleic acid of the present invention, a signal peptide encoded bya nucleic acid of the present invention, or any other polypeptide of thepresent invention. Therefore, the epitope(s) or epitope bearingpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or otherwise described herein. Therefore, the presentinvention includes antibodies that specifically bind specifiedpolypeptides of the present invention, and allows for the exclusion ofthe same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not specificallybind any other analog, ortholog, or homolog of the polypeptides of thepresent invention are included. Antibodies that do not bind polypeptideswith less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55%,and less than 50% identity (as calculated using methods known in the artand described herein, eg., using FASTDB and the parameters set forthherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies, which only bind polypeptides encoded by polynucleotides,which hybridize to a polynucleotide of the present invention understringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms oftheir binding affinity. Preferred binding affinities include those witha dissociation constant or Kd value less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples (See, e.g., Harlow et al., 1988).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,antibodies of the present invention may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “antibody” refers to apolypeptide or group of polypeptides which are comprised of at least onebinding domain, where a binding domain is formed from the folding ofvariable domains of an antibody molecule to form three-dimensionalbinding spaces with an internal surface shape and charge distributioncomplementary to the features of an antigenic determinant of an antigen,which allows an immunological reaction with the antigen. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including eukaryotic, prokaryotic, or phage clone, and notthe method by which it is produced. Monoclonal antibodies can beprepared using a wide variety of techniques known in the art includingthe use of hybridoma, recombinant, and phage display technology.

Hybridoma techniques include those known in the art (See, e.g., Harlowet al. 1988); Hammerling, et al, 1981) (said references incorporated byreference in their entireties). Fab and F(ab′)2 fragments may beproduced, for example, from hybridoma-produced antibodies by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle, whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface orbead., Phage used in these methods are typically filamentous phageincluding fd and M13 with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995); Ames, R. S. et al. (1995); Kettleborough, C. A. et al.(1994); Persic, L. et al. (1997); Burton, D. R. et al. (1994);PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′F(ab)2 and F(ab′)2 fragments can also be employed using methods known inthe art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.(1992); and Sawai, H. et al. (1995); and Better, M. et al. (1988).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991); Shu, L. et al. (1993); and Skerra, A.et al. (1988). For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, (1985); Oi et al.,(1986); Gillies, S. D. et al. (1989); and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596;Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al.,1994), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above. See also, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art (See e.g., Harbor etal. supra; WO 93/21232; EP 0 439 095; Naramura, M. et al. 1994; U.S.Pat. No. 5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al.,1991).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivohalf-life of the polypeptides or for use in immunoassays using methodsknown in the art. The polypeptides may also be fused or conjugated tothe above antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851; 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991); Zheng, X. X. et al. (1995); and Vil, H. et al. (1992).

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies that disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies, which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies that bind the ligand and prevent binding of theligand to the receptor, as well as antibodies that bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies that activatethe receptor. These antibodies may act as agonists for either all orless than all of the biological activities affected by ligand-mediatedreceptor activation. The antibodies may be specified as agonists orantagonists for biological activities comprising specific activitiesdisclosed herein. The above antibody agonists can be made using methodsknown in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng,B. et al. (1998); Chen, Z. et al. (1998); Harrop, J. A. et al. (1998);Zhu, Z. et al. (1998); Yoon, D. Y. et al. (1998); Prat, M. et al. (1998)J.; Pitard, V. et al. (1997); Liautard, J. et al. (1997); Carlson, N. G.et al. (1997) J.; Taryman, R. E. et al. (1995); Muller, Y. A. et al.(1998); Bartunek, P. et al. (1996).

As discussed above, antibodies of the polypeptides of the invention can,in turn, be utilized to generate anti-idiotypic antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art (See, e.g. Greenspan and Bona (1989); and Nissinoff(1991). For example, antibodies which bind to and competitively inhibitpolypeptide multimerization or binding of a polypeptide of the inventionto ligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization or binding domain and, as a consequence,bind to and neutralize polypeptide or its ligand. Such neutralizationanti-idiotypic antibodies can be used to bind a polypeptide of theinvention or to bind its ligands/receptors, and therby block itsbiological activity,

The invention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated full length or mature polypeptide ofthe present invention or to a fragment or variant thereof comprising anepitope of the mutated polypeptide. In another preferred embodiment, thepresent invention concerns an antibody capable of binding to apolypeptide comprising at least 10 consecutive amino acids of apolypeptide of the present invention and including at least one of theamino acids which can be encoded by the trait causing mutations.

Non-human animals or mammals, whether wild-type or transgenic, whichexpress a different species of a polypeptide of the present inventionthan the one to which antibody binding is desired, and animals which donot express a polypeptide of the present invention (i.e. a knockoutanimal) are particularly useful for preparing antibodies. Gene knock outanimals will recognize all or most of the exposed regions of apolypeptide of the present invention as foreign antigens, and thereforeproduce antibodies with a wider array of epitopes. Moreover, smallerpolypeptides with only 10 to 30 amino acids may be useful in obtainingspecific binding to any one of the polypeptides of the presentinvention. In addition, the humoral immune system of animals thatproduce a species of a polypeptide of the present invention thatresembles the antigenic sequence will preferentially recognize thedifferences between the animal's native polypeptide species and theantigen sequence, and produce antibodies to these unique sites in theantigen sequence. Such a technique will be particularly useful inobtaining antibodies that specifically bind to any one of thepolypeptides of the present invention.

Antibody preparations prepared according to either protocol are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

The antibodies of the invention may be labelled by any one of theradioactive, fluorescent or enzymatic labels known in the art.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a polypeptide of the present inventionaccording to the invention in a biological sample, said methodcomprising the following steps:

-   -   a) obtaining a biological sample suspected of containing a        polypeptide of the present invention;    -   b) contacting the biological sample with a polyclonal or        monoclonal antibody that specifically binds a polypeptide of the        present invention under conditions suitable for antigen-antibody        binding; and    -   c) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro thepresence of a polypeptide of the present invention in a biologicalsample, wherein said kit comprises:

-   -   a) a polyclonal or monoclonal antibody that specifically binds a        polypeptide of the present invention, optionally labelled;    -   b) a reagent allowing the detection of the antigen-antibody        complexes formed, said reagent carrying optionally a label, or        being able to be recognized itself by a labelled reagent, more        particularly in the case when the above-mentioned monoclonal or        polyclonal antibody is not labelled by itself.        A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of any of the peptides identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler, G. and Milstein, C., Nature 256:495(1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein or peptidesderived therefrom over a period of a few weeks. The mouse is thensacrificed, and the antibody producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as Elisa,as originally described by Engvall, E., Meth Enzymol 70:419 (1980), andderivative methods thereof. Selected positive clones can be expanded andtheir monoclonal antibody product harvested for use. Detailed proceduresfor monoclonal antibody production are described in Davis, L. et al.Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom described above, whichcan be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and may require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).

Antibody preparations prepared according to either protocol are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

XIII. Identifying One or More Cell Types Expressing a Cell SurfaceReceptor for KRIB Polypeptide

The invention features methods of identifying one or more cell typesexpressing a cell surface receptor for KRIB polypeptide comprised ofcontacting said cell type with labelled KRIB polypeptide and measuringthe amount of said polypeptide bound.

Preferably said labelled KRIB polypeptide is selected from but notrestricted to fluorescein-coupled KRIB or biotin-coupled KRIB. Boundfluorescein-coupled KRIB is detected directly by FACS. Boundbiotin-coupled KRIB is detected by FACS after secondary binding ofphycoerythrin-coupled streptavidin or by radioassay after secondarybinding of ¹²⁵I-streptavidin. Alternatively, KRIB polypeptide is taggedwith an antibody epitope at the N- or C-terminus as described supra withregard to polynucleotides encoding polypeptides of the invention thatare fused in frame to the coding sequences for additional heterologousamino acid sequences. Binding of said epitope-tagged KRIB polypeptide isdetected with antibody specific for the epitope.

XIV. Cloning cDNA Encoding Cell Surface Receptor for KRIB Polypeptide

The invention features methods of using KRIB polypeptide to clone cDNAencoding a cell surface receptor for said KRIB polypeptide. Preferablysaid KRIB polypeptide has activity selected from the group consisting oflipid partitioning, lipid metabolism, and insulin-like activities. Alsopreferably said KRIB polypeptide has activity selected from the groupconsisting of prevention of weight gain, weight reduction, andmaintenance of weight loss.

In a preferred embodiment, said method of cloning a cell surfacereceptor for KRIB polypeptide comprises: isolating mRNA from a cell typeexpressing said cell surface receptor for KRIB polypeptide; convertingsaid mRNA to cDNA; ligating said cDNA into a eukaryotic expressionvector containing the origin for SV40 replication; transientlytransfecting pools of said ligated cDNA into COS cells using dextransulfate; culturing the transfected COS cells for about 48 h; detectingcell surface expression of said receptor for KRIB polypeptide bycontacting said transfected COS cells with biotinylated orepitope-tagged KRIB polypeptide; contacting said biotinylated orepitope-tagged KRIB polypeptide bound to said transfected COS cellsdirectly (biotinylated said polypeptide) or indirectly (epitope-taggedsaid polypeptide) with ¹²⁵I-streptavidin; identifying said transfectedCOS cells labelled with ¹²⁵I-streptavidin; recovering cDNA from saidlabelled COS cells; and repeating said transient transfection withsmaller pools of said recovered cDNA until transfection with a singleclone of cDNA leads to cell surface expression of said receptor for KRIBpolypeptide. Said method of cloning cDNA encoding a cell surfacereceptor for KRIB polypeptide by transient transfection of COS cells iswell known to those skilled in the art.

Other characteristics and advantages of the invention are described inthe Examples. These are meant to be exemplary only, and not to limit theinvention in any way. Throughout this application, various publications,patents and published patent applications are cited. The disclosures ofthese publications, patents and published patent specificationsreferenced in this application are hereby incorporated by reference intothe present disclosure.

EXAMPLES

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein allof which form part of the instant invention.

Example 1 Northern Analysis of KRIB mRNA

Analysis of KRIB expression in different human tissues (adult and fetal)and cell lines, as well as mouse embryos in different stages ofdevelopment, is accomplished by using poly A⁺ RNA blots purchased fromClontech (e.g. #7780-1, 7757-1, 7756-1, 7768-1 and 7763-1). Labeling ofRNA probes is performed using the RNA Strip-EZ kit from Ambion as permanufacture's instructions. Hybridization of RNA probes to RNA blots isperformed Ultrahyb hybridization solution (Ambion). Briefly, blots areprehybridized for 30 min at 58° C. (low-strigency) or 65° C. (highstringency). After adding the labelled probe (2×10⁶ cpm/ml), blots arehybridized overnight (14-24 hrs), and washed 2×20 min at 50° C. with2×SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1×SSC/0.1% SDS(medium stringency) and 2×20 min at 65° C. with 1×SSC/0.1% SDS (highstringency). After washings are completed blots are exposed on thephosphoimager (Molecular Dynamics) for 1-3 days.

Example 2 In Vitro Tests of Metabolic Activity

The activity of various preparations and various sequence variants ofKRIB polypeptides are assessed using various in vitro assays includingthose provided below. These assays are also exemplary of those that canbe used to develop KRIB polypeptide antagonists and agonists. To dothat, the effect of KRIB polypeptides in the above assays, e.g. onleptin and/or LSR activity, in the presence of the candidate moleculeswould be compared with the effect of KRIB polypeptides in the assays inthe absence of the candidate molecules. Since KRIB polypeptides reducebody weight in mice on a high-cafeteria diet (Example 5), these assaysalso serve to identify candidate treatments for reducing (or increasing)body weight.

Liver Cell Line:

Tests of efficacy of KRIB polypeptides on LSR can be performed usingliver cell lines, including for example, PLC, HepG2, Hep3B (human), Hepa1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat).

BPRCL mouse liver cells (ATCC Repository) are plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media is changed on day 2. On day 3, the confluent monolayers are washedonce with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well). Cells areincubated at 37° C. for 30 min with increasing concentrations ofrecombinant KRIB polypeptide or KRIB polypeptide fragment in DMEMcontaining 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl₂, 3.7 g/L sodiumbicarbonate, pH 7.5. Incubations are continued for 3 h at 37° C. afteraddition of 10 ng/mL ¹²⁵I-mouse leptin (specific activity, 22100cpm/ng). Monolayers are washed 2 times consecutively with PBS containing0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 timesconsecutively with PBS. Cells are lysed with 0.1 N NaOH containing 0.24mM EDTA. Lysates are collected into tubes, and counted in agamma-counter.

Blood Brain Barrier Model:

The effect of KRIB polypeptides on leptin transport in the brain can bedetermined using brain-derived cells. One method that is envisioned isto use the blood/brain barrier model described by Dehouck, et al (JNeurochem 54:1798-801, 1990; hereby incorporated herein by reference inits entirety including any figures, tables, or drawings) that uses aco-culture of brain capillary endothelial cells and astrocytes to testthe effects of KRIB polypeptides on leptin (or other molecules)transport via LSR or other receptors.

This assay would be an indicator of the potential effect of KRIBpolypeptides on leptin transport to the brain and could be used toscreen KRIB polypeptide variants for their ability to modulate leptintransport through LSR or other receptors in the brain. In addition,putative agonists and antagonists of the effect of KRIB polypeptides onleptin transport through LSR or other receptors could also be screenedusing this assay. Increased transport of leptin across the blood/brainbarrier would presumably increase its action as a satiety factor.

FACS Analysis of LSR Expression

The effect of KRIB polypeptides on LSR can also be determined bymeasuring the level of LSR expression at the cell surface by flowsurface cytometry, using anti-LSR antibodies and fluorescent secondaryantibodies. Flow cytometry is a laser-based technology that is used tomeasure characteristics of biological particles. The underlyingprinciple of flow cytometry is that light is scattered and fluorescenceis emitted as light from the excitation source strikes the movingparticles.

This is a high throughput assay that could be easily adapted to screenKRIB polypeptides and variants as well as putative agonists orantagonists of KRIB polypeptides. Two assays are provided below. Theantibody, cell-line and KRIB polypeptide analogs would vary depending onthe experiment, but a human cell-line, human anti-LSR antibody and KRIBpolypeptide fragment could be used to screen for variants, agonists, andantagonists to be used to treat humans.

Assay 1:

Cells are pretreated with either intact KRIB polypeptide or KRIBpolypeptide fragment (or untreated) before harvesting and analysis byFACS. Cells are harvested using non-enzymatic dissociation solution(Sigma), and then are incubated for 1 h at 4° C. with a 1:200 dilutionof anti-LSR 81B or an irrelevant anti-serum in PBS containing 1% (w/v)BSA. After washing twice with the same buffer, goat anti-rabbitFITC-conjugated antibody (Rockland, Gilbertsville, Pa.) is added to thecells, followed by a further incubation for 30 min at 4° C. Afterwashing, the cells are fixed in 2% formalin. Flow cytometry analysis isdone on a FACSCalibur cytometer (Becton-Dickinson, Franklin Lakes,N.J.).

Assay 2:

Cells are cultured in T175 flasks according to manufacturer'sinstructions for 48 hours prior to analysis.

Cells are washed once with FACS buffer (1×PBS/2% FBS, filtersterilized), and manually scraped from the flask in 10 mLs of FACSbuffer. The cell suspension is transferred to a 15 mL conical tube andcentrifuged at 1200 rpm, 4° C. for 5 minutes. Supernatant is discardedand cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cellcount is performed and the cell density adjusted with FACS buffer to aconcentration of 1×10⁶ cells/mL. One milliliter of cell suspension wasadded to each well of a 48 well plate for analysis. Cells arecentrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked toensure that cells are pelleted, the supernatant is removed and cellsresuspended by running plate over a vortex mixer. One milliliter of FACSbuffer is added to each well, followed by centrifugation at 1200 rpm for5 minutes at 4° C. This described cell washing was performed a total of3 times.

Primary antibody, titered in screening experiments to determine properworking dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500,1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells ina total volume of 50 μL FACS buffer. Plates are incubated for 1 h at 4°C. protected from light. Following incubation, cells are washed 3 timesas directed above. Appropriate secondary antibody, titered in screeningexperiments to determine proper working dilutions (for example 1:25,1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000,or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.Plates are incubated for 1 h at 4° C. protected from light. Followingincubation, cells are washed 3 times as directed above. Upon final wash,cells are resuspended in 500 μL FACS buffer and transferred to a FACSacquisition tube. Samples are placed on ice protected from light andanalyzed within 1 hour.

Cellular Binding and Uptake of KRIB Polypeptides as Detected byFluorescence Microscopy

Fluorecein isothiocyanate (FITC) conjugation of KRIB polypeptides:Purified KRIB proteins at 1 mg/mL concentration are labelled with FITCusing Sigma's FluoroTag FITC conjugation kit (Stock No. FITC-1).Protocol outlined in the Sigma Handbook for small-scale conjugation isfollowed for KRIB protein labeling.

Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va.CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830)are seeded into 6 well plates at a cell density of 2×10⁵ cells per well.C2C12 and Hepa-1-6 cells are cultured according to repository'sinstructions for 24-48 hours prior to analysis. Assay is performed whencells were 80% confluent.

FITC labelled KRIB protein cellular binding and uptake using microscopy:C2C12 and Hepa 1-6 cells are incubated in the presence/absence ofantibody directed against human LSR (81B: N-terminal sequence of humanLSR; does not cross react with mouse LSR and 93A: c-terminal sequence,cross reacts with mouse LSR) or an antiserum directed against gC1qr(953) for 1 hour at 37° C., 5% CO₂. LSR antibodies are added to themedia at a concentration of 2 μg/mL. The anti-gC1qr antiserum is addedto the media at a volume of 2.5 μL undiluted serum (high concentration)or 1:100 dilution (low concentration). Following incubation withspecified antibody, FITC-KRIB polypeptide (50 nM/mL) is added to eachcell culture well. Cells, are again incubated for 1 hour at 37° C., 5%CO₂. Cells are washed 2× with PBS, cells are scraped from well into 1 mLof PBS. Cell suspension is transferred to an eppendorf tube andcentrifuged at 1000 rpm for 2 minutes. Supernatant is removed and cellsresuspended in 200 μL of PBS. Binding and uptake of FITC-KRIBpolypeptide is analyzed by fluorescence microscopy under 40×magnification.

This assay may be useful for identifying agents that facilitate orprevent the uptake and/or binding of KRIB polypeptides to cells.

Effect on LSR as a Lipoprotein Receptor

The effect of KRIB protein on the lipoprotein binding, internalizing anddegrading activity of LSR can also be tested. Measurement of LSR aslipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry31:4628-36; hereby incorporated herein in its entirety including anydrawings, tables, or figures). The effect of KRIB protein on thelipoprotein binding, internalizing and degrading activity of LSR (orother receptors) can be compared with that of intact KRIB protein, withuntreated cells as an additional control. This assay can also be used toscreen for active and inhibitory variants of KRIB protein, as well asagonists and antagonists of metabolic activity.

Human liver PLC cells (ATCC Repository) are plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media is changed on day 2. On day 3, the confluent monolayers are washedonce with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well). Cells areincubated at 37° C. for 30 min with 10 ng/mL human recombinant leptin inDMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl₂, 3.7 μL sodiumbicarbonate, pH 7.5, followed by another 30 min incubation at 37° C.with increasing concentrations of KRIB polypeptide. Incubations arecontinued for 2 h at 37° C. after addition of 0.8 mM oleate and 20 μg/mL¹²⁵I-LDL. Monolayers are washed 2 times consecutively with PBScontaining 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 timesconsecutively with PBS. The amounts of oleate-induced binding, uptakeand degradation of ¹²⁵I-LDL are measured as previously described (Bihain& Yen, 1992, supra). Results are shown as the mean of triplicatedeterminations.

KRIB protein leads to an increased activity of LSR as a lipoproteinreceptor. The oleate-induced binding and uptake of LDL would be moreaffected by KRIB protein as compared to the degradation. This increasedLSR activity would potentially result in an enhanced clearance oftriglyceride-rich lipoproteins during the postprandial state. Thus, moredietary fat would be removed through the liver, rather than beingdeposited in the adipose tissue.

This assay could be used to determine the efficiency of a compound (oragonists or antagonists) to increase or decrease LSR activity (orlipoprotein uptake, binding and degradation through other receptors),and thus affect the rate of clearance of triglyceride-rich lipoproteins.

Effect on Muscle Differentiation

C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville,Md.) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine,pen/strep, etc)+10% FCS. Two days later they become 80-90% confluent. Atthis time, the media is changed to DMEM+2% horse serum to allowdifferentiation. The media is changed daily. Abundant myotube formationoccurs after 34 days of being in 2% horse serum, although the exact timecourse of C2C12 differentiation depends on how long they have beenpassaged and how they have been maintained, among other things.

To test the effect of the presence of KRIB protein on muscledifferentiation, KRIB polypeptide or polypeptide fragment (1 to 2.5μg/mL) is added the day after seeding when the cells are still in DMEMw/ 10% FCS. Two days after plating the cells (one day after said KRIBpolypeptide or polypeptide fragment was first added), at about 80-90%confluency, the media is changed to DMEM+2% horse serum plus said KRIBpolypeptide or polypeptide fragment.

Effect on Muscle Cell Fatty Acid Oxidation

C2C12 cells are differentiated in the presence or absence of 2 μg/mLKRIB protein for 4 days. On day 4, oleate oxidation rates are determinedby measuring conversion of 1-¹⁴C-oleate (0.2 mM) to ¹⁴CO₂ for 90 min.This experiment can be used to screen for active polypeptides andpeptides as well as agonists and antagonists or activators andinhibitors of KRIB polypeptides.

The effect of KRIB polypeptide or polypeptide fragment on the rate ofoleate oxidation can be compared in differentiated C2C12 cells (murineskeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocytecell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830). Cultured cells aremaintained according to manufacturer's instructions. The oleateoxidation assay is performed as previously described (Muoio et al (1999)Biochem J 338; 783-791). Briefly, nearly confluent myocytes are kept inlow serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, atwhich time formation of myotubes became maximal. Hepatocytes are kept inthe same DMEM medium supplemented with 10% FCS for 2 days. One hourprior to the experiment the media is removed and 1 mL of preincubationmedia (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 nM Hepes,1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added. At thestart of the oxidation experiment ¹⁴C-Oleic acid (1 μCi/mL, AmericanRadiolabelled Chemical Inc., St. Louis, Mo.) is added and cells areincubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL KRIBpolypeptide or polypeptide fragment. After the incubation period 0.75 mLof the media is removed and assayed for ¹⁴C-oxidation products asdescribed below for the muscle FFA oxidation experiment.

Triglyceride and Protein Analysis following Oleate Oxidation in CulturedCells

Following transfer of media for oleate oxidation assay, cells are placedon ice. To determine triglyceride and protein content, cells are washedwith 1 mL of 1×PBS to remove residual media. To each well 300 μL of celldissociation solution (Sigma) is added and incubated at 37° C. for 10min. Plates are tapped to loosen cells, and 0.5 mL of 1×PBS was added.The cell suspension is transferred to an eppendorf tube, each well isrinsed with an additional 0.5 mL of 1×PBS, and is transferred toappropriate eppendorf tube. Samples are centrifuged at 1000 rpm for 10minutes at room temperature. Supernatant is discarded and 750 μL of1×PBS/2% chaps is added to cell pellet. Cell suspension is vortexed andplaced on ice for 1 hour. Samples are then centrifuged at 13000 rpm for20 min at 4° C. Supernatants are transferred to new tube and frozen at−20° C. until analyzed. Quantitative measure of triglyceride level ineach sample is determined using Sigma Diagnostics GPO-TRINDER enzymatickit. The procedure outlined in the manual is adhered to, with thefollowing exceptions: assay is performed in 48 well plate, 350 μL ofsample volume was assayed, control blank consisted of 350 μL PBS/2%CHAPS, and standard contained 10 μL standard provide in kit plus 690 μLPBS/2% chaps. Analysis of samples is carried out on a Packard SpectraCount at a wavelength of 550 nm. Protein analysis is carried out on 25μL of each supernatant sample using the BCA protein assay (Pierce)following manufacturer's instructions. Analysis of samples is carriedout on a Packard Spectra Count at a wavelength of 550 nm.

In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H]-2-deoxyglucose (2DG) with or without KRIB polypeptidefragment in the presence or absence of insulin (10⁻⁸ M) as has beenpreviously described (Walker, P. S. et al. (1990) Glucose transportactivity in L6 muscle cells is regulated by the coordinate control ofsubcellular glucose transporter distribution, biosynthesis, and mRNAtranscription. JBC 265(3):1516-1523; and Kilp, A. et al. (1992)Stimulation of hexose transport by metformin in L6 muscle cells inculture. Endocrinology 130(5):2535-2544, which disclosures are herebyincorporated by reference in their entireties). Uptake of 2DG isexpressed as the percentage change compared with control (no addedinsulin or KRIB polypeptide fragment). Values are presented as mean±SEMof sets of 4 wells per experiment. Differences between sets of wells areevaluated by Student's t test, probability values p<0.05 are consideredto be significant.

Example 3 Effect of KRIB Polypeptides on Mice Fed a High-Fat Diet

Experiments are performed using approximately 6 week old C57B1/6 mice (8per group). All mice are housed individually. The mice are maintained ona high fat diet throughout each experiment. The high fat diet (cafeteriadiet; D12331 from Research Diets, Inc.) has the following composition:protein kcal % 16, sucrose kcal % 26, and fat kcal % 58. The fat isprimarily composed of coconut oil, hydrogenated.

After the mice are fed a high fat diet for 6 days, micro-osmotic pumpsare inserted using isoflurane anesthesia, and are used to providefull-length KRIB polypeptides, KRIB polypeptide fragments, saline, andan irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.KRIB polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/dayand the irrelevant peptide is provided at 10 μg/day. Body weight ismeasured on the first, third and fifth day of the high fat diet, andthen daily after the start of treatment. Final blood samples are takenby cardiac puncture and are used to determine triglyceride (TG), totalcholesterol (TC), glucose, leptin, and insulin levels. The amount offood consumed per day is also determined for each group.

Example 4 Tests of Metabolic Activity in Humans

Tests of the efficacy of KRIB polypeptides in humans are performed inaccordance with a physician's recommendations and with establishedguidelines. The parameters tested in mice are also tested in humans(e.g. food intake, weight, TG, TC, glucose, insulin, leptin, FFA). It isexpected that the physiological factors would show changes over theshort term. Changes in weight gain might require a longer period oftime. In addition, the diet would need to be carefully monitored. KRIBpolypeptides, preferably KRIB polypeptides, would be given in dailydoses of about 6 mg protein per 70 kg person or about 10 mg per day.Other doses would also be tested, for instance 1 mg or 5 mg per day upto 20 mg, 50 mg, or 100 mg per day.

Example 5 Tests of Metabolic Activity in a Murine Lipoatrophic DiabetesModel

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401:73-76 (1999); hereby incorporated herein in its entiretyincluding any drawings, figures, or tables). Leptin was found to be lesseffective in a different lipodystrophic mouse model of lipoatrophicdiabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporatedherein in its entirety including any drawings, figures, or tables). Theinstant invention encompasses the use of KRIB polypeptides for reducingthe insulin resistance and hyperglycaemia in this model either alone orin combination with leptin, the leptin peptide (U.S. provisionalapplication No. 60/155,506), or other compounds. Assays include thatdescribed previously in Gavrilova et al. ((2000) Diabetes 49:1910-6;(2000) Nature 403:850) using A-ZIP/F-1 mice, except that KRIBpolypeptides would be administered using the methods previouslydescribed in Example 3 (or Examples 6-8). The glucose and insulin levelsof the mice would be tested, and the food intake and liver weightmonitored, as well as other factors, such as leptin, FFA, and TG levels,typically measured in our experiments (see Example 3, above, or Examples6-8).

Example 6 Effect of KRIB Polypeptides on Plasma Free Fatty Acid in C57BL/6 Mice

The effect of KRIB polypeptides on postprandial lipemia (PPL) in normalC57BL6/J mice is tested.

The mice used in this experiment are fasted for 2 hours prior to theexperiment after which a baseline blood sample is taken. All bloodsamples are taken from the tail using EDTA coated capillary tubes (50 μLeach time point). At time 0 (8:30 AM), a standard high fat meal (6 gbutter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mLdistilled water prepared fresh following Nb#6, JF, pg. 1) is given bygavage (vol.=1% of body weight) to all animals.

Immediately following the high fat meal, 25 μg a KRIB polypeptide isinjected i.p. in 100 μL saline. The same dose (25 μg/mL in 100 μL) isagain injected at 45 min and at 1 hr 45 min. Control animals areinjected with saline (3×100 μL). Untreated and treated animals arehandled in an alternating mode.

Blood samples are taken in hourly intervals, and are immediately put onice. Plasma is prepared by centrifugation following each time point.Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG)and glucose are determined within 24 hours using standard test kits(Sigma and Wako). Due to the limited amount of plasma available, glucoseis determined in duplicate using pooled samples. For each time point,equal volumes of plasma from all 8 animals per treatment group arepooled.

Example 7 Effect of KRIB Polypeptides on Plasma Leptin and Insulin inC57 BL/6 Mice

The effect of KRIB polypeptides on plasma leptin and insulin levelsduring postprandial lipemia (PPL) in normal C57BL6/J mice is tested. Theexperimental procedure is the same as that described in Example 6,except that blood was drawn only at 0, 2 and 4 hours to allow forgreater blood samples needed for the determination of leptin and insulinby RIA.

Briefly, 16 mice are fasted for 2 hours prior to the experiment afterwhich a baseline blood sample is taken. All blood samples are taken fromthe tail using EDTA coated capillary tubes (100 μL each time point). Attime 0 (9:00 AM), a standard high fat meal (see Example 6) is given bygavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 25 μg of a KRIB polypeptide is injected i.p. in 100μL saline. The same dose (25 μg in 100 μL) is again injected at 45 minand at 1 hr 45 min (treated group). Control animals are injected withsaline (3×100 μL). Untreated and treated animals are handled in analternating mode.

Blood samples are immediately put on ice and plasma is prepared bycentrifugation following each time point. Plasma is kept at −20° C. andfree fatty acids (FFA) are determined within 24 hours using a standardtest kit (Wako). Leptin and Insulin are determined by RIA (ML-82K andSR1-13K, LINCO Research, Inc., St. Charles, Mo.) following themanufacturer's protocol; however, only 20 μL plasma is used. Eachdetermination is done in duplicate. Due to the limited amount of plasmaavailable, leptin and insulin are determined in 4 pools of 2 animalseach in both treatment groups.

Example 8 Effect of KRIB Polypeptides on Plasma FFA, TG and Glucose inC57 BL/6 Mice

The effect of KRIB polypeptides on plasma FFA, TG, glucose, leptin andinsulin levels during postprandial lipemia (PPL) in normal C57BL6/J micehas been described. Weight loss resulting from KRIB polypeptides (2.5μg/day) given to normal C57BL6/J mice on a high fat diet has also beenshown (Example 3).

The experimental procedure is similar to that described in Example 6.Briefly, 14 mice re fasted for 2 hours prior to the experiment afterwhich a baseline blood sample is taken. All blood samples are taken fromthe tail using EDTA coated capillary tubes (50 μL each time point). Attime 0 (9:00 AM), a standard high fat meal (see Example 6) is given bygavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 4 mice are injected 25 μg of a KRIB polypeptide i.p.in 100 μL saline. The same dose (25 μg in 100 μL) is again injected at45 min and at 1 hr 45 min. A second treatment group receives 3 times 50μg KRIB polypeptide at the same intervals. Control animals are injectedwith saline (3×100 μL). Untreated and treated animals are handled in analternating mode.

Blood samples are immediately put on ice. Plasma is prepared bycentrifugation following each time point. Plasma is kept at −20° C. andfree fatty acids (FFA), triglycerides (TG) and glucose are determinedwithin 24 hours using standard test kits (Sigma and Wako).

Example 9 Effect of KRIB Polypeptides on FFA Following EpinephrineInjection

In mice, plasma free fatty acids increase after intragastricadministration of a high fat/sucrose test meal. These free fatty acidsare mostly produced by the activity of lipolytic enzymes i.e.lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, theseenzymes are found in significant amounts both bound to endothelium andfreely circulating in plasma. Another source of plasma free fatty acidsis hormone sensitive lipase (HSL) that releases free fatty acids fromadipose tissue after β-adrenergic stimulation. To test whether KRIBpolypeptides also regulate the metabolism of free fatty acid released byHSL, mice are injected with epinephrine.

Two groups of mice are given epinephrine (5 μg) by intraperitonealinjection. A treated group is injected with a KRIB polypeptide (25 μg)one hour before and again together with epinephrine, while controlanimals receive saline. Plasma is isolated and free fatty acids andglucose are measured as described above (Example 8).

Example 10 Effect of KRIB Polypeptides on Muscle FFA Oxidation

To investigate the effect of KRIB polypeptides on muscle free fatty acidoxidation, intact hind limb muscles from C57BL16J mice are isolated andFFA oxidation is measured using oleate as substrate (Clee, S. M. et al.Plasma and vessel wall lipoprotein lipase have different roles inatherosclerosis. J Lipid Res 41, 521-531 (2000); Muoio, D. M., Dohm, G.L., Tapscott, E. B. & Coleman, R. A. Leptin opposes insulin's effects onfatty acid partitioning in muscles isolated from obese ob/ob mice. Am JPhysiol 276, E913-921 (1999)) Oleate oxidation in isolated muscle ismeasured as previously described (Cuendet et al (1976) J Clin Invest58:1078-1088; Le Marchand-Brustel, Y., Jeanrenaud, B. & Freychet, P.Insulin binding and effects in isolated soleus muscle of lean and obesemice. Am J Physiol 234, E348-E358 (1978). Briefly, mice are sacrificedby cervical dislocation and soleus and EDL muscles are rapidly isolatedfrom the hind limbs. The distal tendon of each muscle is tied to a pieceof suture to facilitate transfer among different media. All incubationsare carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonatebuffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 2.54mM CaCl₂, 25 mM NaHCO₃, 10 mM Hepes, pH 7.4) supplemented with 4% FFAfree bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mMglucose (Sigma). The total concentration of oleate (Sigma) throughoutthe experiment is 0.25 mM. All media are oxygenated (95% O₂; 5% CO₂)prior to incubation. The gas mixture is hydrated throughout theexperiment by bubbling through a gas washer (Kontes Inc., Vineland,N.J.).

Muscles are rinsed for 30 min in incubation media with oxygenation. Themuscles are then transferred to fresh media (1.5 mL) and incubated at30° C. in the presence of 1 μCi/mL [1-¹⁴C] oleic acid (AmericanRadiolabelled Chemicals). The incubation vials containing this media aresealed with a rubber septum from which a center well carrying a piece ofWhatman paper (1.5 cm×11.5 cm) is suspended.

After an initial incubation period of 10 min with constant oxygenation,gas circulation is removed to close the system to the outsideenvironment and the muscles are incubated for 90 min at 30° C. At theend of this period, 0.45 μL of Solvable (Packard Instruments, Meriden,Conn.) is injected onto the Whatman paper in the center well and oleateoxidation by the muscle is stopped by transferring the vial onto ice.

After 5 min, the muscle is removed from the medium, and an aliquot of0.5 mL medium is also removed. The vials are closed again and 1 mL of35% perchloric acid is injected with a syringe into the media bypiercing through the rubber septum. The CO₂ released from the acidifiedmedia is collected by the Solvable in the center well. After a 90 mincollection period at 30° C., the Whatman paper is removed from thecenter well and placed in scintillation vials containing 15 mL ofscintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).The amount of ¹⁴C radioactivity is quantitated by liquid scintillationcounting. The rate of oleate oxidation is expressed as nmol oleateproduced in 90 min/g muscle.

To test the effect of full-length KRIB polypeptide or KRIB polypeptidefragment on oleate oxidation, these proteins are added to the media at afinal concentration of 2.5 μg/mL and maintained in the media throughoutthe procedure.

Example 11 Effect of KRIB Polypeptides on Triglyceride in Muscle & LiverIsolated from Mice

To determine whether the increased FFA oxidation induced by KRIBpolypeptides is also accompanied by increased FFA delivery into muscleor liver, the hindlimb muscle and liver triglyceride content is measuredafter the KRIB polypeptide treatment of mice. Hind limb muscles as wellas liver samples are removed from treated and untreated animals and thetriglyceride and free fatty acid concentration is determined following astandard lipid extraction method (Shimabukuro, M. et al. Directantidiabetic effect of leptin through triglyceride depletion of tissues.Proc Natl Acad Sci USA 94:4637-4641 (1997)) followed by TG and FFAanalysis using standard test kits.

Example 12 Effect of KRIB Polypeptides on FFA Following IntralipidInjection

Two groups of mice are intravenously (tail vein) injected with 30 μLbolus of Intralipid-20% (Clintec) to generate a sudden rise in plasmaFFAs, thus by-passing intestinal absorption. (Intralipid is anintravenous fat emulsion used in nutritional therapy). A treated group(KRIB polypeptide-treated) is injected with a KRIB polypeptide (25 μg)at 30 and 60 minutes before Intralipid is given, while control animalsreceive saline. Plasma is isolated and FFAs are measured as describedpreviously. The effect of KRIB polypeptides on the decay in plasma FFAsfollowing the peak induced by Intralipid injection is then monitored.

Example 13 In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H]-2-deoxyglucose (2DG) with or without KRIB polypeptides in thepresence or absence of insulin (10⁻⁸ M) as has been previously described(Walker, P. S. et al. (1990) Glucose transport activity in L6 musclecells is regulated by the coordinate control of subcellular glucosetransporter distribution, biosynthesis, and mRNA transcription. JBC265:1516-1523; and Kilp, A. et al. (1992) Stimulation of hexosetransport by metformin in L6 muscle cells in culture, Endocrinology130:2535-2544, which disclosures are hereby incorporated by reference intheir entireties). Uptake of 2DG is expressed as the percentage changecompared with control (no added insulin or KRIB). Values are presentedas mean±SEM of sets of 4 wells per experiment. Differences between setsof wells are evaluated by Student's t test, probability values p<0.05are considered to be significant.

Example 14 In Vivo Tests for Metabolic Activity in Rodent DiabetesModels

As metabolic profiles differ among various animal models of obesity anddiabetes, analysis of multiple models is undertaken to separate theeffects KRIB polypeptides on hyperglycemia, hyperinsulinemia,hyperlipidemia and obesity. Mutations within colonies of laboratoryanimals and different sensitivities to dietary regimens have made thedevelopment of animal models with non-insulin dependent diabetesassociated with obesity and insulin resistance possible. Genetic modelssuch as db/db and ob/ob (See Diabetes, (1982) 31(1): -6) in mice andfa/fa in zucker rats have been developed by the various laboratories forunderstanding the pathophysiology of disorder and testing the efficacyof new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu RepSankyo Res Lab (1994) 46: 1-57). The homozygous animals, C57BL/KsJ-dbldb mice developed by Jackson Laboratory, US, are obese,hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest,(1990) 85: 962-967), whereas heterozygous are lean and normoglycemic. Indb/db model, mouse progressively develops insulinopenia with age, afeature commonly observed in late stages of human type II diabetes whenblood sugar levels are insufficiently controlled. The state of pancreasand its course vary according to the models. Since this model resemblesthat of type II diabetes mellitus, the compounds of the presentinvention are tested for blood sugar and triglycerides loweringactivities. Zucker (fa/fa) rats are severely obese, hyperinsulinemic,and insulin resistant (Coleman, Diabetes 31;1, 1982; E. Shafrir, inDiabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier SciencePublishing Co., Inc., New York, ed. 4, 1990), pp. 299-340), and thefa/fa mutation may be the rat equivalent of the murine db mutation(Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc. Natl.Acad. Sci. USA 88:7806, 1991). Tubby (tub/tub) mice are characterized byobesity, moderate insulin resistance and hyperinsulinemia withoutsignificant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401: 73-76 (1999). Leptin is found to be less effective in adifferent lipodystrophic mouse model of lipoatrophic diabetes (Gavrilovaet al Nature 403: 850 (2000); hereby incorporated herein in its entiretyincluding any drawings, figures, or tables).

The streptozotocin (STZ) model for chemically-induced diabetes is testedto examine the effects of hyperglycemia in the absence of obesity.STZ-treated animals are deficient in insulin and severely hyperglycemic(Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H.Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc.,New York, ed. 4, 1990), pp. 299-340). The monosodium glutamate (MSG)model for chemically-induced obesity (Olney, Science 164:719, 1969;Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesityis less severe than in the genetic models and develops withouthyperphagia, hyperinsulinemia and insulin resistance, is also examined.Finally, a non-chemical, non-genetic model for induction of obesityincludes feeding rodents a high fat/high carbohydrate (cafeteria diet)diet ad libitum.

The instant invention encompasses the use of KRIB polypeptides forreducing the insulin resistance and hyperglycemia in any or all of theabove rodent diabetes models or in humans with Type I or Type IIdiabetes or other prefered metabolic disorders described previously ormodels based on other mammals. In the compositions of the presentinvention the KRIB polypeptides may, if desired, be associated withother compatible pharmacologically active antidiabetic agents such asinsulin, leptin (U.S. provisional application No. 60/155,506), ortroglitazone, either alone or in combination. Assays include thatdescribed previously in Gavrilova et al. ((2000) Diabetes 49:1910-6;(2000) Nature 403:850) using A-ZIP/F-1 mice, except that KRIBpolypeptides are administered intraperitoneally, subcutaneously,intramuscularly or intravenously. The glucose and insulin levels of themice would be tested, and the food intake and liver weight monitored, aswell as other factors, such as leptin, FFA, and TG levels, typicallymeasured in our experiments.

In Vivo Assay for Anti-Hyperglycemic Activity of KRIB Polypeptides

Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old)are housed (7-9 mice/cage) under standard laboratory conditions at 22°C. and 50% relative humidity, and maintained on a diet of Purina rodentchow and water ad libitum. Prior to treatment, blood is collected fromthe tail vein of each animal and blood glucose concentrations aredetermined using One Touch Basic Glucose Monitor System (Lifescan). Micethat have plasma glucose levels between 250 to 500 mg/dl are used. Eachtreatment group consists of seven mice that are distributed so that themean glucose levels are equivalent in each group at the start of thestudy db/db mice are dosed by micro-osmotic pumps, inserted usingisoflurane anesthesia, to provide KRIB polypeptides, saline, and anirrelevant peptide to the mice subcutaneously (s.c.). Blood is sampledfrom the tail vein hourly for 4 hours and at 24, 30 h post-dosing andanalyzed for blood glucose concentrations. Food is withdrawn from 0-4 hpost dosing and reintroduced thereafter. Individual body weights andmean food consumption (each cage) are also measured after 24 h.Significant differences between groups (comparing KRIB treated tosaline-treated) are evaluated using Student t-test.

In Vivo Insulin Sensitivity Assay

In vivo insulin sensitivity is examined by utilizing two-stephyperinsulinemic-euglycemic clamps according to the following protocol.Rodents from any or all of the various models described in Example 2 arehoused for at least a week prior to experimental procedures. Surgeriesfor the placement of jugular vein and carotid artery catheters areperformed under sterile conditions using ketamine and xylazine (i.m.)anesthesia. After surgery, all rodents are allowed to regainconsciousness and placed in individual cages. KRIB polypeptides orvehicle is administered through the jugular vein after complete recoveryand for the following two days. Sixteen hours after the last treatment,hyperinsulinemic-euglycemic clamps are performed. Rodents are placed inrestrainers and a bolus of 4 μCi [3-³H] glucose (NEN) is administered,followed by a continuous infusion of the tracer at a dose of 0.2μCi/min. (20 μl/min). Two hours after the start of the tracer infusion,3 blood samples (0.3 ml each) are collected at 10 minute intervals(−20-0 min) for basal measurements. An insulin infusion is then started(5 mU/kg/min), and 100 μl blood samples are taken every 10 min. tomonitor plasma glucose. A 30% glucose solution is infused using a secondpump based on the plasma glucose levels in order to reach and maintaineuglycemia. Once a steady state is established at 5 mU/kg/min insulin(stable glucose infusion rate and plasma glucose), 3 additional bloodsamples (0.3 ml each) are obtained for measurements of glucose, [3-³H]glucose and insulin (100-120 min.). A higher dose of insulin (25mU/kg/min.) is then administered and glucose infusion rates are adjustedfor the second euglycemic clamp and blood samples are taken at min.220-240. Glucose specific activity is determined in deproteinized plasmaand the calculations of Rd and hepatic glucose output (HGO) are made, asdescribed (Lang et al., Endocrinology 130:43, 1992). Plasma insulinlevels at basal period and after 5 and 25 mU/kg/min. infusions are thendetermined and compared between KRIB treated and vehicle treatedrodents.

Insulin regulation of glucose homeostasis has two major components;stimulation of peripheral glucose uptake and suppression of hepaticglucose output. Using tracer studies in the glucose clamps, it ispossible to determine which portion of the insulin response is affectedby the KRIB polypeptides.

Example 15 Effect of KRIB Polypeptides on Weight Gain and Weight Loss ofMice and on Maintenance of Weight Loss in Mice

In the first experiment, 10-week-old male C57BL/6J mice are put on avery high fat/sucrose purified diet for 19 days to promote weight gain(see Example 3); the average body weight at this time is 30 g. The miceare then surgically implanted with an osmotic pump (Alzet, Newark, Del.)delivering either 2.5 μg/day of KRIB polypeptide fragment, 5 μg/day offull-length KRIB, or physiological saline. The mice are continued on thehigh fat diet and their body weight was recorded over the following10-day period.

Weight gain by mice treated with saline in contradistinction to weightloss by mice treated either with KRIB polypeptide fragment or KRIBfull-length polypeptide is taken as evidence that in this inbred strainof normal mice, a continuous infusion of a daily low dose of KRIBpolypeptide fragment or KRIB full-length polypeptide can prevent weightgain caused by high fat/sucrose feeding, in a sustainable way.

Data are expressed throughout as mean±SEM; a p-value <0.05 is consideredstatistically significant. Statistical analysis is typically done usingeither the unpaired Student's t test or the paired Student's t test.

Maintenance of Weight Loss in Mice

In order to demonstrate the ability of KRIB polypeptide to maintainweight loss, normal mice are put on a reduced calorie diet to promoteweight loss. The reduced calorie diet is continued until the mice lose10% of their initial weight. A second group of mice are continued on thereduced calorie diet until the mice lose 20% of their initial weight.The mice are then surgically implanted with an osmotic pump (Alzet,Newark, Del.) delivering either 2.5 μg/day of KRIB polypeptide fragment,5 μg/day of KRIB full-length polypeptide, or physiological saline. Themice are returned to a normal diet and their body weights are recordedover a 10-day period. After 10 days, the outcome wherein mice treatedwith KRIB polypeptide fragment or KRIB full-length polypeptide have alower weight than mice treated with saline is taken to provide evidencethat treatment with KRIB polypeptide fragment or KRIB full-lengthpolypeptide promotes the maintenance of weight loss.

Example 16 Effect of KRIB Polypeptides on the Phosphorylation State ofProtein Kinase C Alpha (PKCα)

Cells are treated with either 5 μg/ml KRIB polypeptide or physiologicalsaline for 5, 10, 30, and 60 min. Cells are then washed and lysed in 50mM Tris pH7.6, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 1 mM EDTA,1:100 Phosphatase Inhibitor Cocktail I and II (Sigma), and Completeprotease inhibitor cocktail (Roche Diagnostics). The amount ofSer657-phosphorylated PKCα was assessed by Western blot analysis usingan affinity-purified PKC antibody that recognizes a conservedhydrophobic C-terminal FXXF(S/T)(F/Y) motif only when theserine/threonine residues are phosphorylated (Cell SignalingTechnology). Total amounts of protein in the individual lanes arenormalized by Western blotting using a monoclonal antibody againstα-tubulin (Sigma).

Example 17 Effect of KRIB Polypeptides on NF-κB Activation

A. Activation of NF-κB by KRIB Polypeptide

Luciferase activity is measured in transfected cells following overnightincubation with 200 ng/ml LPS (E. coli serotype 055:B5, Sigma) or 5μg/ml KRIB polypeptide before and after proteinase K and heat treatment.Cells are transfected with an E-selectin promoter-luciferase construct[Schindler U et al. (1994) Mol Cell Biol Sep; 14(9):5820-31, which ishereby incorporated by reference in its entirety] and CMVpromoter-α-galactosidase as internal transfection efficiency controlusing FuGene 6 reagent (Roche) according to manufacturer's instructions.Incubation is performed in normal growth media. To test for endotoxincontamination, KRIB polypeptide and LPS are treated with proteinase K(0.2 mg/ml) for 90 min at 50° C., followed by 12 min at 99° C.Resistance of LPS and sensitivity of KRIB polypeptide to digestionestablishes that activities of the latter are not due to contaminationby bacterial endotoxin.

B. Ser32 Phosphorylation and Degradation of IκB-α by KRIB Polypeptide

Cells are treated with 200 ng/ml LPS or 5 μg/ml KRIB polypeptide innormal growth media. After 30 and 120 min of incubation, cells arewashed with PBS containing Ca²⁺ and Mg²⁺ and lysed as in Example 16. Theamount of phosphorylated and total IκB-α in lysates is assessed byWestern blot analysis using affinity purified Ser32 phospho-specificantibody and a different phosphorylation state-independent IκB-αantibody (Cell Signaling Technology).

Example 18 Effect of iAFLP Gene Expression Profiling Analysis of Poly A+and Total RNA from Different Tissue Sources

Introduced Amplified Fragment Length Polymorphism (iAFLP) methodology isadapted from an article entitled “Expression Profiling by iAFLP: A PCRbased Method for Genome Wide Gene Expression Profiling” (Kawamoto etal., Genome Research. 9:1305-1312, 1999).

IAFLP gene expression is preformed using Poly A+ RNA and Total RNA fromdifferent adult human tissues purchased from Ambion (#7961-Liver,7967-Heart, 7963-Brain, 7983-Skeletal Muscle, 7951-Placenta, and7951-Small Intestine) and Research Genetics (#D6005-01-Adipose Tissue),respectively. Total RNA is DNase treated for 30 minutes at 37 C. 20 ugof total RNA or 2 ug of Poly A+ RNA is converted to dscDNA using thecDNA Synthesis System supplied by Roche Applied Science as per themanufacture's instructions. The purified and quantified dscDNA iscleaved using the MboI restriction enzyme kit supplied by New EnglandBiolabs as per the manufacture's instructions. One-third of the MboIcleaved dscDNA is ligated to kinated adaptor cassette primers using theT4 DNA Ligation kit supplied by Roche Applied Science as per themanufacture's instructions. The ligated dscDNA is diluted in glycogenand DI water to a final concentration of 1 ng/ul. One ng is added to afinal PCR master mix volume of 10 ul containing 0.2 mM dNTPs, 1 mM PCRBuffer, 2 mM MgCl₂, 2 uM Vic fluorescent labeled T7 primer, 2 uM of agene-specific reverse primer, 8% glycerol, and 1 Unit Amplitaq Gold DNAPolymerase. The template is incubated for 10 min at 95 C, denatured for30 sec. at 95 C, annealed for 1 min at 60 C, extended for 30 sec. at 72C for 35 cycles and extended 7 min at 72 C for 1 cycle.

PCR reaction is performed using the Applied Biosystem's 9700 GeneAmpthermalcycler. One ul of the PCR reaction is diluted 1:100. 1 ul of thediluted PCR product is combined with 0.1 ul of the Liz 500 size standard(Applied Biosystems) and 8.9 ul of HiDi Formamide (Applied Biosystems).The mixture is denatured for 5 min. at 95 C. 1 ul of denatured mixtureis loaded into a 3700 DNA Analyzer (Applied Biosystems) and separated bysize. Analysis of the differently sized fragments is performed by theGenescan software package supplied by Applied Biosystems as per themanufacture's instructions.

Example 19 iAFLP Gene Expression Profiling Analysis of KRIBpolynucleotides

The BodyMap site discloses results obtained using the iAFLP technology.In the frame of the present invention, it has been found thatnucleotides 425 to 631 of SEQ ID NO: 1 (KRIB-1) are 99% identical toBodyMap Accession No. GS13677, which is expressed 5.7-fold in adiposetissue compared with a mix of tissues. Nucleotides 206 to 431 of SEQ IDNO: 3 (KRIB-2) are 90% identical to nucleotides 1 to 231 of GS03123, andnucleotides 83 to 191 of SEQ ID NO: 5 (KRIB-2R) are 99% identical tonucleotides 1 to 110 of GS03123. GS03123 is expressed 9.4-fold inadipose tissue compared with a mix of tissues. Nucleotides 1742 to 1986of SEQ ID NO:7 (KRIB-3) are 98.8% identical to BodyMap Accession No.GS00237, which is expressed 8.1-fold in adipose tissue compared with amix of tissues. Nucleotides 1361 to 1415 are 80% identical tonucleotides 1 to 55 of ap3439, which corresponds to BodyMap AccessionNo. GS03136. GS03136 is expressed 5.9-fold in adipose tissue comparedwith a mix of tissues (BodyMap Accession No. GS03136). Thus KRIB-1,KRIB-2, KRIB-2R, KRIB-3 and KRIB-4 are all expressed at higher levels inadipose tissue than in other tissues, rendering them therapeuticproteins for treatment of obesity-related disorders.

1-15. (canceled)
 16. A method for treating a metabolic disorder selectedfrom the group consisting of obesity, impaired glucose tolerance,insulin resistance, Syndrome X, and Type II diabetes comprising theadministration of a KRIB polypeptide is selected from the groupconsisting of: (a) a KRIB-1 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 2; (b) a KRIB-1 polypeptide comprising SEQ ID NO: 2;(c) a KRIB-1 polypeptide comprising amino acids 20 to 136 of SEQ ID NO:2; (d) a variant of (b) or (c), wherein the amino acid sequence has atleast 50% or 60% or 70% or 80% or 90% identity to at least one of thesequences in (b) or (c); (e) a variant of (b) or (c) which is encoded bya DNA sequence which hybridizes to the complement of the DNA sequenceencoding (b) or (c) under moderately stringent conditions or underhighly stringent conditions; (f) a variant of (b) or (c) wherein anychanges in the amino acid sequence are conservative amino acidsubstitutions to the amino acid sequences in (b) or (c); (g) a KRIB-2polypeptide comprising at least 6 amino acids of SEQ ID NO: 4; (h) aKRIB-2 polypeptide comprising SEQ ID NO: 4; (i) a KRIB-2 polypeptidecomprising amino acids 15 to 97 of SEQ ID NO: 4; (j) a KRIB-2polypeptide comprising the LAWN Domain region at amino acids positions72 to 94 of SEQ ID NO: 4; (k) a variant of any of (h) to (j), whereinthe amino acid sequence has at least 50% or 60% or 70% or 80% or 90%identity to at least one of the sequences in (h) to (j); (l) a variantof any of (h) to (j) which is encoded by a DNA sequence which hybridizesto the complement of the DNA sequence encoding any of (h) to (j) undermoderately stringent conditions or under highly stringent conditions;(m) a variant of any of (h) to (j) wherein any changes in the amino acidsequence are conservative amino acid substitutions to the amino acidsequences in (h) to (j); (n) a KRIB-2R polypeptide comprising at least 6amino acids of SEQ ID NO: 6; (o) a KRIB-2R polypeptide comprising SEQ IDNO: 6; (p) a KRIB-2R polypeptide comprising amino acids 15 to 111 of SEQID NO: 6; (q) a KRIB-2R polypeptide comprising the LAWN Domain region atamino acids positions 39 to 61 of SEQ ID NO: 6; (r) a variant of any of(o) to (q), wherein the amino acid sequence has at least 50% or 60% or70% or 80% or 90% identity to at least one of the sequences in (o) to(q); (s) a variant of any of (o) to (q) which is encoded by a DNAsequence which hybridizes to the complement of the DNA sequence encodingany of (o) to (q) under moderately stringent conditions or under highlystringent conditions; (t) a variant of any of (o) to (q) wherein anychanges in the amino acid sequence are conservative amino acidsubstitutions to the amino acid sequences in (o) to (q); (u) a KRIB-3polypeptide comprising at least 6 amino acids of SEQ ID NO: 8; (v) aKRIB-3 polypeptide comprising SEQ ID NO: 8; (w) a KRIB-3 polypeptidecomprising amino acids 18 to 212 of SEQ ID NO: 8; (x) a variant of (v)or (w), wherein the amino acid sequence has at least 50% or 60% or 70%or 80% or 90% identity to at least one of the sequences in (v) or (w);(y) a variant of (v) or (w) which is encoded by a DNA sequence whichhybridizes to the complement of the DNA sequence encoding (v) or (w)under moderately stringent conditions or under highly stringentconditions; (z) a variant of (v) or (w) wherein any changes in the aminoacid sequence are conservative amino acid substitutions to the aminoacid sequences in (v) or (w); (aa) a KRIB-4 polypeptide comprising atleast 6 amino acids of SEQ ID NO: 10; (bb) a KRIB-4 polypeptidecomprising SEQ ID NO: 10; (cc) a KRIB-4 polypeptide comprising aminoacids 36 to 347 of SEQ ID NO: 10; (dd) a polypeptide comprising one ofthe leucine rich repeat domains at amino acid positions 93 to 116, 117to 140, 141 to 164, 165 to 188, 189 to 212, 213 to 236, 237 to 260 and261 to 284 of SEQ ID NO: 10; (ee) a polypeptide comprising one of thesegments exhibiting a periodic pattern in the occurrence of leucine,proline, and asparagines at amino acid positions 84 to 107, 109 to 131,132 to 155, 156 to 179, 180 to 203, 204 to 227, 228 to 251 and 252 to275 of SEQ ID NO: 10; (ff) a polypeptide comprising the leucine richrepeat C-terminal domain at amino acid positions 299 to 347 of SEQ IDNO: 10; (gg) a variant of any of (bb) to (ff), wherein the amino acidsequence has at least 50% or 60% or 70% or 80% or 90% identity to atleast one of the sequences in (bb) to (ff); (hh) a variant of any of(bb) to (ff) which is encoded by a DNA sequence which hybridizes to thecomplement of the DNA sequence encoding any of (bb) to (ff) undermoderately stringent conditions or under highly stringent conditions;and (ii) a variant of any of (bb) to (ff) wherein any changes in theamino acid sequence are conservative amino acid substitutions to theamino acid sequences in (bb) to (ff).
 17. A method of screening for KRIBligands comprising: (a) contacting a KRIB polypeptide with a testcompound; and (b) determining whether said compound specifically bindsto said polypeptide, wherein a detection that said compound specificallybinds to said polypeptide indicates that said compound is a ligand ofsaid KRIB polypeptide.
 18. The method according to claim 17, whereinsaid KRIB polypeptide is selected from the group consisting of: (a) aKRIB-1 polypeptide comprising at least 6 amino acids of SEQ ID NO: 2;(b) a KRIB-1 polypeptide comprising SEQ ID NO: 2; (c) a KRIB-1polypeptide comprising amino acids 20 to 136 of SEQ ID NO: 2; (d) avariant of (b) or (c), wherein the amino acid sequence has at least 50%or 60% or 70% or 80% or 90% identity to at least one of the sequences in(b) or (c); (e) a variant of (b) or (c) which is encoded by a DNAsequence which hybridizes to the complement of the DNA sequence encoding(b) or (c) under moderately stringent conditions or under highlystringent conditions; (f) a variant of (b) or (c) wherein any changes inthe amino acid sequence are conservative amino acid substitutions to theamino acid sequences in (b) or (c); (g) a KRIB-2 polypeptide comprisingat least 6 amino acids of SEQ ID NO: 4; (h) a KRIB-2 polypeptidecomprising SEQ ID NO: 4; (i) a KRIB-2 polypeptide comprising amino acids15 to 97 of SEQ ID NO: 4; (j) a KRIB-2 polypeptide comprising the LAWNDomain region at amino acids positions 72 to 94 of SEQ ID NO: 4; (k) avariant of any of (h) to (j), wherein the amino acid sequence has atleast 50% or 60% or 70% or 80% or 90% identity to at least one of thesequences in (h) to (j); (l) a variant of any of (h) to (j) which isencoded by a DNA sequence which hybridizes to the complement of the DNAsequence encoding any of (h) to (j) under moderately stringentconditions or under highly stringent conditions; (m) a variant of any of(h) to (j) wherein any changes in the amino acid sequence areconservative amino acid substitutions to the amino acid sequences in (h)to (j); (n) a KRIB-2R polypeptide comprising at least 6 amino acids ofSEQ ID NO: 6; (o) a KRIB-2R polypeptide comprising SEQ ID NO: 6; (p) aKRIB-2R polypeptide comprising amino acids 15 to 111 of SEQ ID NO: 6;(q) a KRIB-2R polypeptide comprising the LAWN Domain region at aminoacids positions 39 to 61 of SEQ ID NO: 6; (r) a variant of any of (o) to(q), wherein the amino acid sequence has at least 50% or 60% or 70% or80% or 90% identity to at least one of the sequences in (o) to (q); (s)a variant of any of (o) to (q) which is encoded by a DNA sequence whichhybridizes to the complement of the DNA sequence encoding any of (o) to(q) under moderately stringent conditions or under highly stringentconditions; (t) a variant of any of (o) to (q) wherein any changes inthe amino acid sequence are conservative amino acid substitutions to theamino acid sequences in (o) to (C); (u) a KRIB-3 polypeptide comprisingat least 6 amino acids of SEQ ID NO: 8; (v) a KRIB-3 polypeptidecomprising SEQ ID NO: 8; (w) a KRIB-3 polypeptide comprising amino acids18 to 212 of SEQ ID NO: 8; (x) a variant of (v) or (w), wherein theamino acid sequence has at least 50% or 60% or 70% or 80% or 90%identity to at least one of the sequences in (v) or (w); (y) a variantof (v) or (w) which is encoded by a DNA sequence which hybridizes to thecomplement of the DNA sequence encoding (v) or (w) under moderatelystringent conditions or under highly stringent conditions; (z) a variantof (v) or (w) wherein any changes in the amino acid sequence areconservative amino acid substitutions to the amino acid sequences in (v)or (w); (aa) a KRIB-4 polypeptide comprising at least 6 amino acids ofSEQ ID NO: 10; (bb) a KRIB-4 polypeptide comprising SEQ ID NO: 10; (cc)a KRIB-4 polypeptide comprising amino acids 36 to 347 of SEQ ID NO: 10;(dd) a polypeptide comprising one of the leucine rich repeat domains atamino acid positions 93 to 116, 117 to 140, 141 to 164, 165 to 188, 189to 212, 213 to 236, 237 to 260 and 261 to 284 of SEQ ID NO: 10; (ee) apolypeptide comprising one of the segments exhibiting a periodic patternin the occurrence of leucine, proline, and asparagines at amino acidpositions 84 to 107, 109 to 131, 132 to 155, 156 to 179, 180 to 203, 204to 227, 228 to 251 and 252 to 275 of SEQ ID NO: 10; (ff) a polypeptidecomprising the leucine rich repeat C-terminal domain at amino acidpositions 299 to 347 of SEQ ID NO: 10; (gg) a variant of any of (bb) to(ff), wherein the amino acid sequence has at least 50% or 60% or 70% or80% or 90% identity to at least one of the sequences in (bb) to (ff);(hh) a variant of any of (bb) to (ff) which is encoded by a DNA sequencewhich hybridizes to the complement of the DNA sequence encoding any of(bb) to (ff) under moderately stringent conditions or under highlystringent conditions; and (ii) a variant of any of (bb) to (ff) whereinany changes in the amino acid sequence are conservative amino acidsubstitutions to the amino acid sequences in (bb) to (ff).
 19. A methodof treating a metabolic disorder selected from the group consisting ofobesity, impaired glucose tolerance, insulin resistance, Syndrome X, andType II diabetes comprising the administration of a KRIB agonist to anindividual.
 20. A method of treating a metabolic disorder selected fromthe group consisting of cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, anorexia, and bulimia comprising theadministration of a KRIB antagonist to an individual.
 21. The methodaccording to claim 20, wherein said KRIB antagonist is an antibody. 22.A method of treating a metabolic disorder selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,Syndrome X, and Type II diabetes comprising the administration of a genetherapy vector comprising a nucleotide sequence encoding a KRIBpolypeptide to an individual, wherein said KRIB polypeptide is selectedfrom the group consisting of: (a) a KRIB-1 polypeptide comprising SEQ IDNO: 2; (b) a KRIB-2 polypeptide comprising SEQ ID NO: 4; (c) a KRIB-2Rpolypeptide comprising SEQ ID NO: 6; (d) a KRIB-3 polypeptide comprisingSEQ ID NO: 8; and (e) a KRIB-4 polypeptide comprising SEQ ID NO:
 10. 23.A method of screening candidate compounds for a KRIB agonist or a KRIBantagonist comprising the steps of: (a) contacting a cell with a KRIBpolypeptide with or without a candidate compound; and (b) detecting theagonist or antagonist activity of said compound on the basis ofactivity, wherein said activity is selected from the group consistingof: LSR expression, leptin transport, increase in glucose uptake,decrease in blood lipid or triglyceride levels, increase in lipoproteinbinding, uptake or degradation, and increase in free fatty acidoxidation.
 24. A method of screening candidate compounds for a KRIBagonist or a KRIB antagonist comprising the steps of: (a) administeringto a test animal a KRIB polypeptide with or without a candidatecompound, wherein said KRIB polypeptide is selected from the groupconsisting of: (i) a KRIB-1 polypeptide comprising at least 6 aminoacids of SEQ ID NO: 2; (ii) a KRIB-2 polypeptide or comprising at least6 amino acids of SEQ ID NO: 4; (iii) a KRIB-2R polypeptide comprising atleast 6 amino acids of or SEQ ID NO: 6; (iv) a KRIB-3 polypeptidecomprising at least 6 amino acids of SEQ ID NO: 8; and (v) a KRIB-4polypeptide comprising at least 6 amino acids of SEQ ID NO: 10; and; (b)detecting the ability of said compound to agonize or antagonize saidKRIB polypeptide on the basis of activity, wherein said activity isselected from the group consisting of: prevention of weight gain, weightreduction, and maintenance of weight loss.